JP2022058602A - Recombinant aav vectors expressing osteoprotective genes, including has2 and lubricin, useful in treatment of osteoarthritis and related joint conditions in mammals - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide recombinant viral vectors, pharmaceutical compositions comprising such recombinant vectors, and methods for prevention and treatment of osteoarthritis in mammals.

SOLUTION: This disclosure provides adeno-associated virus (AAV) vectors capable of expressing, in a host, osteoprotective/chondroprotective bioactive proteins, including hyaluronan synthase 2 (HAS2) and lubricin (PRG4). Methods of production of these AAVs are provided, as are methods of treatment of osteoarthritis in mammalian joints, by the long-term gene expression of osteoprotective/chondroprotective proteins, including HAS2 and PRG4, in both synovial and chondrocyte cells.

SELECTED DRAWING: None

COPYRIGHT: (C)2022,JPO&INPIT

Description

(Mutual Citing of Related Applications) This application claims priority to US Provisional Patent Application No. 62 / 278,243 (filed January 13, 2016, which is incorporated herein by reference in its entirety).
(Description of Sequence Listing) The sequence listing accompanying this application is provided in text form on behalf of the printout copy, which is incorporated herein by reference. The name of the text file containing the sequence listing is MER 16-291 SEQ Listing_ST25.txt. The text file is 57.6KB and was created on January 13, 2016. The above is submitted electronically by EFS-Web at the same time as the filing of this specification.
(Technical field)
The present invention relates to recombinant vectors, pharmaceutical compositions containing such recombinant vectors, and methods for the prevention and / or treatment of acute and / or chronic joint symptoms (including osteoarthritis) in animals. In particular, the present invention relates to an adeno-associated virus (AAV) vector capable of expressing a bioactive polypeptide belonging to the hyaluronan synthase 2 (HAS2) and lubricin (PRG4) family proteins in a host. Accordingly, the present invention relates to adeno-associated virus (AAV), which is used in the field of genetic manipulation for the treatment of osteoarthritis of human or mammalian joints by long-term gene expression of HAS2 and LUB in synovial and cartilage cells. Provide a base biological delivery and expression system.

Osteoarthritis (OA) is a degenerative joint disease that occurs in mammalian joints and causes serious economic and medical problems (Matthews, GL, and Hunter, DJ (2011). Expert Opin. Emerging Drugs 1- 13; Brooks PM.Curr Opin Rheumatol 2002; 14: 573-577). Cartilage is a strong connective tissue that covers the epiphysis with joints. The above provides a highly lubricated surface with relatively low friction between hard bones, providing smooth movement. During OA progression, cartilage is partially or completely lost due to abnormal or excessive wear, leading to epiphyseal exposure, which rub against each other, resulting in inflammation, pain, swelling or decreased mobility. At present, the exact reason for the early cartilage loss leading to OA is unknown, but there is a strong correlation between its occurrence and age, obesity and joint abuse (eg, athletic activity).
In dogs, osteoarthritis (OA) is one of the most common causes of repellency, and it is estimated that about 20 percent of dogs over the age of 1 year will be affected. At present, there are no curative treatments available for OA, so medical procedures are largely directed at symptom relief rather than cartilage regeneration. Analgesic treatments usually include steroids and non-steroidal anti-inflammatory drugs (NSAIDS), which have been effective in the treatment of OA for decades. However, although these agents can suppress joint inflammation, many of them are known to have degenerative effects on cartilage (this effect further exacerbates the underlying process of OA progression). In addition to traditional analgesic and anti-inflammatory therapies, direct administration of naturally occurring bone-protecting compounds has been used to relieve OA symptoms, but to varying degrees. For example, hyaluronic acid (HA) has been widely used to restore viscoelasticity and lubricity of affected joints. Furthermore, polysulfated glycosaminoglycans (PSGAG) (injected by the intra-articular or intramuscular route) and orally administered glucosamine and chondroitin sulfate have shown some efficacy.

However, the agents mentioned above must be administered frequently (sometimes in combination with each other) to achieve meaningful symptom relief. These frequent joint injections are laborious / costly, pose a risk of infection and cause great stress to the patient or animal. Surgical approaches have also been developed, but they are generally less effective in dogs and horses and are typically performed only in advanced critical target animals.
In addition to supplement / drug delivery, several groups can express in the host a means of producing a viscoelastic / viscoelastically protected polypeptide, a nucleic acid encoding the above, or a viscoelastically protected protein (eg, an enzyme). Attempts were made to ameliorate OA symptoms by delivering polypeptides or nucleic acids. Approaches of greater interest include the use of Lubricin polypeptide (Flannery, US 7,642,236 B2), tribonectin (US 7,618,914 B2 (Rhode Island General Hospital)), and hyaluronan synthase (US 6,423,514 (Millennium Pharmaceuticals)). ..
Some of these attempts can be characterized as "gene therapy" (its basic concept is well established) (Evans CH, Robbins PD.Gene therapy for arthritis, In: Wolff JA (ed.). Gene. Therapeutics: Methods and Applications of Direct Gene Transfer. Birkhauser: Boston, 1994, pp 320-343). Recently, a group attempted to treat osteoarthritis by in vivo delivery of the interleukin-1 receptor antagonist (Il-1Ra) gene (US 2015/0031083 A1 (Baylor College of Medicine); Frisbie, DD et. al., Gene Therapy, 2002).

Arthrogen company expressed human interferon beta (due to inflammatory cytokine lowering) using AAV5 in the context of rheumatoid arthritis (RA). Unlike OA, inflammatory signaling plays an important role in the pathology of RA, so blocking this signal is an important therapeutic approach. However, another group pursuing a possible link between inflammation and OA used recombinant AAV2 to express IL1 receptor antagonists in the case of horse OA (Goodrich et al., Molecular). Therapy-Nucleic Acids (2013) 2, e70).

However, neither of these approaches has been proven to be universally effective, and there are still unsatisfied and serious demands for pain and pain relief in OA patients. As a result, there are clear and yet unfulfilled medical demands for more effective and sustained treatments that are at the same time and cost-effective in the long run.
Therefore, as detailed herein, Applicants have received a cDNA in which a recombinant adeno-associated virus (rAAV) vector encodes a therapeutic agent by a single intra-articular injection into a mammalian joint. For the first time, we have succeeded in delivering and presenting that the substance can promote local and continuous in vivo production in synovial and chondrocytes.
For the first time, Applicants also isolated and sequenced the full-length inulbricin cDNA (SEQ ID NO: 4).

The present invention provides an rAAV vector that expresses a therapeutically effective amount of a bone protection gene and / or a bone regeneration gene product in vivo in a mammalian host.
In multiple features, the rAAV can include cDNA encoding a substance with disease-correcting, lubricating, anti-inflammatory and pain-relieving properties.

In multiple features, the rAAV vector is derived from an AAV serotype containing, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8 and AAVrh.10. It is a vector. In some embodiments, the nucleic acid of AAV comprises an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV2rAAV6, AAV7, AAV8, AAV9, AAVrh.8 or AAVrh.10. In yet another embodiment, the rAAV particles include a capsid protein of AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8 or AAVrh.10. In some embodiments, the ITR and capsid are derived from the same AAV serotype. In other embodiments, the ITR and capsid are derived from different AAV serotypes. In some embodiments, the rAAV vector may be of the AAV2 or AAV5 capsid serotype. In some related embodiments, the rAAV2 and rAAV5 vectors contain at least one ITR derived from AAV2.
In embodiments of multiple features, the rAAV vector encodes hyaluronate synthase-2 (HAS2) or a variant thereof. In some embodiments, the HAS2 is human HAS2. In another embodiment, the HAS2 is a dog HAS2. In some embodiments, HAS2 is a codon-optimized HAS2.
Glycosaminoglycan hyaluronan acid (HA) is a non-sulfated glycosaminoglycan and consists of repetitive glucuronic acid and N-acetylglucosamine residues linked by beta-1-3 and beta-1-4 glycosidic bonds. HA provides multiple biological functions, including wound healing, cell migration, malignant transformation and tissue turnover. HA is synthesized by a variety of cell types, including endothelial cells, fibroblasts and smooth muscle cells, and has been detected in tissues such as connective tissue, epithelial tissue and neural tissue. In the inter-articular space, HA is produced by chondrocytes along with synovial cells (which secrete HA into synovial fluid). Synovial fluid HA plays a role in joint homeostasis as well as providing a scaffold for lubrication, tissue hydration, structural integrity, and matrix proteins and biological mechanisms. The biological action of HA in joints is determined by its concentration and molecular weight. HA can be synthesized in the range of 5,000 Da to 10,000,000 Da. The smaller molecular weight HA (<500 kDa) is closely associated with receptor-mediated activation of angiogenesis, malignancy and inflammation, while the higher molecular weight HA provides lubrication in the joints.

Both the size and concentration of joint HA are regulated by their rate of synthesis and degradation. The degradation of HA is mediated by hyaluronidase, which cleaves HA into smaller fragments, which are excreted into the lymphatic system for removal. Three enzymes (referred to as hyaluronan synthases (HAS) 1, 2 and 3) have been described for HA production, which are present on the inner surface of the synovial cell plasma membrane. Of these, HAS2 has been shown to be required for the production of high molecular weight HA (Itano et al., 1999). High molecular weight HA levels are reduced in both human and animal OA models of osteoarthritis (Plickert et al., 2013). This is probably due to a decrease in HA synthesis and an increase in HA degradation due to hyaluronidase. Of the HA synthases, HAS2 and 3 are expressed in human cartilage, of which HAS2 expression is reduced in human OA. HA levels also decrease due to increased levels of the HA-degrading enzyme hyaluronidase 2 (Yoshida et al.). The HAS2 promoter has been reported to be responsive to a variety of pre- and anti-inflammatory mediators and has been reported to have conflicting effects. These mediators include TGFβ, primary epidermal growth factor, TNFalpha and retinoic acid (Guo, Kanter el al., 2007; Hyc et al., 2009). Downregulation by inflammation mediators in diseased joints is expected to reduce HAS2 expression, resulting in lower HA levels and differentially affect a variety of HAS isoforms (David-Raoudi et al., 2009). ). In contrast, mechanical stimulation (Momberger et al. 2005) or cartilage components (eg chondroitin sulfate) have been reported to stimulate HA production (Momberger et al., 2005; David-Raoudi et al., 2009). The production of HA results in its pericellular presence as well as its secretion into the extracellular space. It is not clear what regulates the degree of secretion. However, typically about 80% of HA is secreted, while the rest remain bound to the producing cells. This cell-bound HA is important for the assembly of matrix proteins, and blocking HAS2 synthesis results in a decrease in the cell-bound matrix and an increased release of proteoglycans (ie agrecans) into the medium, synthesizing HA in chondrocytes. Further confirm the major role of HAS2 as a major enzyme (Nishida et al., 1999).

In embodiments with multiple features, the rAAV vector encodes lubricin or a variant thereof. In some embodiments, the lubricin is human lubricin. In another embodiment, the rubricin is inulbricin. In some embodiments, the rubricin is a codon-optimized rubricin.
Lubricin (PRG4) is a large mucin glycoprotein produced by articular synovial lining cells and chondrocytes of cartilage, which provides a lubrication that protects the cartilage surface (Flannery 1999; Schmidt 2001; Waller 2013). Lubricin, along with HA, is also an important lubricant in synovial fluid and provides shock absorption properties. The lack of rubricin in mouse models and rare hereditary diseases in humans results in cartilage degeneration characteristic of osteoarthritis (OA) (Rhee 2005; Ruan 2013). Decreased synthesis of lubricin has also been shown in human OA patients and diverse animal OA models (Elsaid, 2008). Intra-articular lubricin replacement with recombinant lubricin has been shown to improve cartilage lesions (Flannery, 2009).
In some embodiments, the rAAV vector may be an AAV2 or AAV5 capsid serotype encoding canine codon-optimized hyaluronate synthase-2 (HAS2).
In multiple features, the rAAV vector is administered by intra-articular delivery. In multiple embodiments, rAAV is administered by a single intra-articular delivery. In another embodiment, following intra-articular administration, in vivo production and secretion of allogeneic therapeutic agents derived from the rAAV transduced cells can be sustained for at least about 6 months.
In some embodiments, the rAAV vector comprises an expression cassette containing a ubiquitous promoter and a codon-optimized species-compatible transgene (see Figure 1A).
In another feature, the disclosure provides a method of in vivo expression of bone protection and / or bone regeneration gene products in animal joints using rAAV vectors.
All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.
The following detailed description (provided by way of example, but not intended to limit the invention to the specific embodiments described) will be best understood in conjunction with the accompanying drawings. The attached drawings are as follows:

FIG. 1A is a schematic diagram showing the HAS expression cassette in the plasmid (upper) and rAAV viral vector (lower). FIG. 1B is a graph showing HA production in cells transfected with the cHAS2 expression plasmid. Three days after transfecting the cHAS expression plasmid into 293 cells, a conditioned culture medium was collected and the HA level in the medium was quantified using a detection system using an HA-binding protein. Abbreviations: CBAcHAS2, HAS2 expression plasmid; EGFP, EGFP expression plasmid; non-transfected, negative control cells; "Optimem" = cells grown in serum-free medium; "Complete", cells grown in serum-containing medium. FIG. 1C is an agarose gel image used for component separation of a conditioned culture medium (24-hour hyaluronidase treatment + or-) of cHAS-transfected 293 cells, confirming HA expression. Abbreviations: CM, conditioned culture; MDa, molecular weight size of marker. FIG. 2A is a graph showing rAAV vector generation by small-scale packaging of cHAS2 into rAAV2 and AAV5 vectors using the triple transfection method. Packaging of the EGFP expression cassette to AAV2 is shown as a positive control and packaging of cHAS in the absence of the capsid plasmid is shown as a negative control. The rAAV yield is expressed as the amount of DNA resistant particles (DRP) per cell. FIG. 2B is a graph showing rAAV vector yield in large-scale vector generation by triple transfection. An example of the total titers obtained for the obtained vector lots is shown. Figure 2C is a graph showing the in vitro performance of the AAV2 / HAS2 vector. 293 cells were infected with various MOIs and HA levels in conditioned culture were quantified after 3 days. Figure 2D is a graph showing the in vitro performance of the AAV5 / HAS2 vector. FIG. 3A is a graph showing body weight changes after intra-articular injection of rAAV / HAS2 vector in normal canine joints. FIG. 3B is a graph showing the cartilage score. FIG. 3C is a graph showing the synovial score. FIG. 4A is a schematic showing sample collection positions for rAAV vector quantification of canine synovium. FIG. 4B is a graph showing the quantification of the number of vector genome copies in synovial sample # 3. FIG. 4C is a graph showing the number of vector genome copies of synovial sample # 1. Vector doses of AAV2 and AAV5 are shown as follows: L = low, M = medium and H = high (similar to FIGS. 3A-C). All tissue samples were collected 28 days after rAAV vector delivery and BGHpA was analyzed by qPCR. FIG. 5A is a graph showing vector-derived cHAS expression in synovial sample # 3. FIG. 5B is a graph showing vector mRNA in synovial sample # 1. FIG. 5C is a graph showing the respective vector genomes and vector-derived mRNAs of individual dogs analyzed using synovial sample # 3. Vector doses of AAV2 and AAV5 are shown as follows: L = low, M = medium and H = high. All tissue samples were collected 28 days after rAAV vector delivery and BGHpA was analyzed by qPCR. FIG. 6A is a schematic showing the location of femoral and tibial plateau samples collected to detect cartilage rAAV vectors and mRNA. FIG. 6B is a graph showing the respective vector genomes and vector-derived mRNAs of individual dogs analyzed using femoral condyle sample # 1. In addition, the vector genome copy count on the contralateral side (non-injected right joint) is shown (excluding sample # 22 (the above was not tested)). FIG. 6C is a graph showing the average of vector genomes (injected and non-injected joints) and mRNA copies of each group. Vector doses of AAV2 and AAV5 are shown as follows: L = low, M = medium and H = high. All tissue samples were collected 28 days after rAAV vector delivery and BGHpA was analyzed by qPCR. FIG. 6D is a graph showing the mean vector genome (PBS or vector injection joint) and mRNA copy in each group of tibial plateaus. FIG. 7A is a graph showing the vector genomes of synovium (samples # 3 and # 1) and cartilage (femoral condyle and tibial plateau) in each treatment group of tissue collected from the left posterior knee joint. The values shown in Figures 7A and 7B represent the mean ± standard deviation of the group (n = 5 / group). FIG. 7B is a graph showing the quantification of vector genomes and mRNAs derived from the rAAV5 / HAS2 vector in diverse tissues. FIG. 8A is a graph showing HA levels in canine synovial fluid. HA levels were quantified in SF samples collected on days-7 (reference line) and day 28. HA levels in each animal were normalized to baseline levels and expressed as% of HA on day 28 compared to the week prior to vector administration. FIG. 8B is a graph showing HA levels in canine synovial fluid on day -7 (reference line) and day 28. Arrows indicate animals with higher HA levels on day 28 compared to the reference line (before treatment). Figure 9 shows the complete amino acid sequence of inulbricin. The framed area indicates the location of exon 6 (mucin domain). Underlined amino acids (378-782) are deleted with abbreviated lubricin (hereinafter “cLub1” or “cLub1co”), and the location of KEPAPTT-like repeats (potential O-linked glycosylation sites) is shown in bold. .. When the name of a sequence ends with "co", it means that the DNA sequence is codon-optimized. Similarly, “nonco” means non-codon optimization. FIG. 10 is a schematic diagram showing a plasmid prepared and used in an experiment. The plasmid contains shortened (ie, internal deletion manipulation), codon-optimized inulbricin sequence (cLub1co), promoter (minCBA or CBA) and BGHpA site. Some constructs include modifications that remove the ATG (potential start codon) from the N- or C-terminal His tag (C-term) and intron sequence. Previrus AAV rubricin also contains flanking ITR sequences at both ends. FIG. 11A is a graph showing mRNA copies / cells produced when minCBA cLub1, CBA CLUB1 and CBH cLub-nonco are transfected into 293 cells. FIG. 11B is a graph showing mRNA copies / cells produced when 293 cells were transfected with ΔATG / 6His / N', 6His / N', 6His / C', WT cLub and EGFP constructs. FIG. 12A is an anti-lubricin western blot showing secreted lubricin levels in concentrated medium (plasmids are described above). Canine synovial fluid was used as a positive control. FIG. 12B is a Western blot showing lubricin production derived from the previral lubricin expression plasmid. Two clones were analyzed and compared to the expression obtained with the minCBA-cLubco plasmid. Non-transfected culture medium and cells transfected with the EGFP expression plasmid were used as negative controls. FIG. 13A is a graph showing vector yields in small-scale vector generation for AAV2 vectors encoding inulbricin. Two cLub clones (-/ + 6xHis tags) were analyzed and compared to the packaging of EGFP and HAS2 expression cassette-carrying previrus (ITR-containing) plasmids. Negative controls included transfections lacking non-transfected cells and the AAV2 capsid expression plasmid. FIG. 13B is a graph showing vector yields in small-scale vector generation for the AAV5 vector encoding inulbricin. Previral plasmids for the EGFP and cLub expression cassettes were transfected with the AAV5 capsid expression plasmid. FIG. 14 is an anti-lubricin western blot showing in vitro expression of inurubricin derived from the rAAV5 vector. Human 293 cells were infected with various amounts of rAAV5 / minCBA-cLub1 for 72 hours, followed by concentration of conditioned culture medium. A culture medium of AAV5 / CBA-EGFP infected cells was used as a negative control. The culture medium of cells transfected with the previrus rubrisin expression plasmid was used as a positive control. FIG. 15 is a table showing the summary of the sequence numbers. Figure 16 shows the alignment of dogs and human rubricin. Figure 16-1 continued. FIG. 17 is a graph showing HA levels at various time points in canine synovial fluid using the MMR model. Synovial fluid was collected 1 week before OA induction (pre), 2 weeks after induction, and before the joint administration test (day 0) and 57, 112, 182 days after the joint delivery test. FIG. 18A is a graph showing the detection and expression of the rAAV5 vector 182 days after the vector administration in the synovial sample of the canine OA joint. FIG. 18B is a graph showing the detection and expression of the rAAV5 vector in the cartilage (femoral condyle) of the canine OA joint 182 days after the vector administration. Figure 18C is a summary of the rAAV5 vector genome and cHAS2 mRNA detection in day 182 synovial and cartilage samples in a canine MMR OA model. FIG. 19 shows safranin-O stained sections of cartilage surface obtained from the inside of PBS- and two rAAV5 / cHAS2-treated dog joints as an example.

Detailed Description of the Invention Osteoarthritis (OA) is one of the most common causes of mammalian and dog repellency, affecting approximately 20% of dogs over 1 year of age. OA is a progressive and degenerative disease that results in pain, inflammation and reduced range of motion. New, safe and effective therapies that improve joint lubrication and reduce inflammation and pain are sought for the management of OA. As disclosed herein, Applicants have only one intra-articular injection of a gene encoding a therapeutic substance with the goal of providing local and continuous production of the substance in the joint. We have found that recombinant adeno-associated virus (AAV) vectors can be used for delivery. The rAAV vector was generated using the AAV2 and AAV5 capsid serotypes and encoded canine codon-optimized hyaluronic acid (HA) synthase-2 (HAS2).
Twenty-two healthy adult dogs (AAV2 and AAV5 capsid serotype negative) were administered rAAV2 (1.5 and 10x10 ¹¹ vg / joint), rAAV5 (5x10 ¹¹ vg / joint) or PBS (control) by intra-articular injection. No adverse clinical signs were observed during the subsequent 28-day study. Histopathological analysis showed minimal synovial inflammation in rAAV5-treated joints and no significant changes in the rAAV2-treated group. Vector genome (VG) was detected in the synovium of all rAAV-treated joints and most cartilage samples. The rAAV5 vector resulted in higher VG detection and mRNA expression in both tissues compared to rAAV2. Preliminary analysis also showed an increasing trend in HA levels in the synovial fluid of the test joint. In summary, our study conducted gene transfer to canine joint tissue and an acceptable safety profile by the rAAV2 and rAAV5 vectors encoding HAS2 when administered in a single intra-articular injection in a limited number of dogs. showed that.

Canine HA synthase 2 : In one feature of the invention, the disclosure provides a recombinant adeno-associated virus (rAAV) vector containing an AAV capsid and a single-stranded DNA genome. The viral capsids of the present disclosure can confer uptake of the vector into joint cells (with subsequent transport into the cell nucleus) and result in expression of the therapeutic agent. In some embodiments, the DNA genome has one or more AAV reverse end repeats (ITRs) flanked into one or more expression cassettes for in vivo expression of the therapeutic gene in an animal host. include. In some embodiments of the invention, the viral gene will be absent or will not be expressed from the rAAV genome.
In some embodiments of the invention, once rAAV is administered to an animal and incorporated into the animal's cells, the rAAV genome will survive as an extrachromosomal episome. In some embodiments, the rAAVs of the present disclosure are applied to joint cells over a long period of time, eg, about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, Can survive for longer than (but not limited to) 11 months, 1 year, 2 years, or 3 years. The episome rAAV will continue to be expressed leading to the production and secretion of the therapeutic substance into the synovial fluid, thereby providing local and continuous production of the substance directly to the joint. The selected transgene products can promote joint health by increasing joint lubricity and reducing pain and cartilage degradation.

In another feature of the invention, the disclosure provides a method of treating an animal in need thereof, said method comprising administering to the animal a therapeutically effective amount of rAAV of the present disclosure. In some embodiments, the method comprises administering to the dog the proposed product directly to the affected joint. In some embodiments, a single treatment is sufficient to affect a significant improvement in animal symptoms.
In other embodiments, the treatment is repeated. In some embodiments, the treatment is repeated within 2-3 weeks of the first dose, in other embodiments the second dose is given more than 3 weeks after the first dose. In some related embodiments, the second dose comprises administration of rAAV with the same therapeutic gene, which rAAV comprises the same serotype capsid as the first treatment. In another related embodiment, the second dose comprises administration of rAAV with the same therapeutic gene, but the rAAV comprises a serotype capsid different from the first administration. In some embodiments, rAAV has a serotype 5 capsid. In a related embodiment (repeated administration is desired), the first dose can include administration of rAAV with a serotype 5 capsid and the second dose can include administration of rAAV with a serotype 5 capsid. Can include.
In multiple features, overexpression of HAS2 protein in osteoarthritis joints increases synovial fluid HA levels and enhances joint health by enhancing HA lubrication, anti-inflammatory and pain-relieving properties. Overexpression of HAS2 has been shown to result in increased HA levels in cultures in vitro by various cell types. The above has been shown as either stable or transient transfection with CHO, 293 and COS cells. To provide in vivo overexpression of HA in joints, HAS2 cDNA can be delivered to cartilage and / or synovium (normal HA synthesis site) by using the rAAV vector encoding the HAS2 expression cassette. Without being bound by any theory, introduction of the gene into the cell will provide sustained expression of HAS2 and subsequent HA production in the HAS2 expression cassette. Since the therapeutic vector will contain a ubiquitous promoter, the vector will not be dominated by downregulation by inflammation mediators present in osteoarthritis joints, unlike the endogenous HAS2 promoter. When a vector is administered by intra-articular injection, the vector can result in transduction of a variety of cell types, the major cell type being synovial cells. Finally, HAS2 alone has been shown to be sufficient for HA synthesis, and other contradictory proteins or components are believed to be unnecessary for HA production in vitro (Yoshida et al.).

Inulbricin : In multiple features, lubricin production in osteoarthritis joints is achieved by intra-articular delivery of a recombinant adeno-associated virus (rAAV) vector that encodes lubricin as a potential treatment for canine osteoarthritis (OA). To increase. Lubricin is a large secreted glycoprotein that acts as a lubricant and protects the cartilage surface of joints. This specification describes the discovery and production of full-length inulbricin cDNA. Using the cDNA, a shortened codon-optimized version of inulbricin (cLub1co) was designed. Subsequently, a variety of rubricin expression plasmids were constructed using the latter (shortened codon-optimized version). After transfection into HEK293 cells, these plasmids were characterized for rubricin mRNA and protein production. The data demonstrated the production of both lubricin mRNA and secreted rubricin from each construct. An rAAV vector was prepared using the cLub1co expression cassette, and the feasibility of rAAV / cLub1 vector generation was shown. This synthetic construct was infected with HEK293 cells to secrete inulbricin.
The methods and compositions described herein can also be used in the therapeutic treatment of osteoarthritis. The terms "therapeutic" or "therapeutic treatment" are either already suffering from osteoarthritis when they relate to osteoarthritis, and when they are used herein and in the veterinary field. Treatment, support and / or accelerated treatment of the subject animal recovering from osteoarthritis (eg, in recovery), or diagnosed with osteoarthritis or at risk of osteoarthritis Regarding treatments aimed at delaying and / or reversing cartilage loss in the subject animal. An important goal of treatment is to reduce the risk of developing cartilage and bone loss. As used herein, if the subject is reasonably expected to suffer from progressive cartilage loss associated with osteoarthritis, the subject is suffering from osteoarthritis or is degenerative. It is said that there is a risk of developing arthritis. Whether or not an individual subject animal has osteoarthritis or is at risk of developing osteoarthritis can be easily determined by the corresponding veterinary or medical expert. ..

The methods and compositions described herein can also be used for the prophylactic treatment of osteoarthritis. The terms "prevention, prophylaxsis" and "preventative treatment, prophylactic treatment" are used when they relate to osteoarthritis and as they are used herein and in the field of human and veterinary medicine. When the subject is healthy or suffering from an unrelated disease, the subject animal is considered to be at risk of osteoarthritis.
Described herein are therapeutic and prophylactic treatments for osteoarthritis, the pharmaceutical composition comprising a vector capable of expressing HAS or lubricin polypeptide in vivo, as well as the pharmaceutical compositions. Utilize methods and compositions that induce a sustained increase in hyaluronic acid or lubricin levels in joints to reduce or eliminate cartilage loss.
As used herein, administration of an amount of the pharmaceutical composition is sufficient to cause a significant improvement in clinical signs or measurable markers of the disease in the subject mammal suffering from osteoarthritis. In some cases, the composition is said to have "therapeutic efficacy" or is said to be "therapeutically effective". As used herein, a composition has "preventive efficacy" if administration of an amount of the pharmaceutical composition is sufficient to prevent the development of osteoarthritis in the subject animal. , Or is said to be "effective".
Also described herein are HAS or lubricin, or vectors capable of in vivo expression of the variants, fragments or combinations described above in a host. In a plurality of embodiments, the HAS or lubricin polypeptide used in the present invention is generally compatible with the target species of interest (eg, a vector encoding canine HAS2 is delivered to dogs suffering from OA).

Examples of "variants", "derivatives", etc. described herein include, but are not limited to, HAS and rubricin variants, derivatives, etc., which are exactly the same as the nucleotide sequences disclosed herein. Not the same as, but changes in the nucleotide sequence do not change the amino acid sequence, or the changes are conservative substitutions of amino acid residues, deletion or addition of one or several amino acids, the said change. Substitution of amino acid residues by an amino acid analog that does not significantly affect the properties of the polypeptide (eg, variants or derivatives are about 10%, 20%, 30%, 40%, 50% of the desired activity of the wild-type polypeptide. It is encoded by a nucleotide sequence that yields%, 60%, 70%, 80%, 90%, 95%, 99% or more activity or 100% activity). Examples of conservative amino acid substitutions are glycine / alanine substitution; valine / isoleucine / leucine substitution; aspartic acid / glutamine substitution; aspartic acid / glutamic acid substitution; serine / threonine / methionine substitution; lysine / arginine substitution; and phenylalanine / tyrosine / tryptophan. Substitution is included. Other types of substitutions, variants, additions, deletions and derivatives that give rise to functional HAS or rubricin derivatives are also described herein and one of ordinary skill in the art will recognize how such variants or derivatives are made. Or it should be screened and it will be readily known how to test the HAS or lubricin activity of such variants or derivatives. One of ordinary skill in the art can optimize the expression of the HAS or lubricin polypeptide of the present invention. For example, removal of hidden splice sites, adaptation of codon frequency by introducing a Kozak consensus sequence before the start codon, modification of codon frequency (but not limited to the above), or a combination of the above improves expression. ..

The vector used in the present invention can include a nucleic acid sequence encoding a canine HAS2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the canine HAS2 polypeptide has SEQ ID NO: 2, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, A canine HAS2 variant with at least 98%, or at least 99% homology or identity.
The vector used in the present invention can include an inulbricin polypeptide comprising the amino acid sequence set set forth in SEQ ID NO: 7. In some embodiments, the inulbricin polypeptide is SEQ ID NO: 7, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, An inulbricin variant having at least 98%, or at least 99% homology or identity.
Sequence identity or homology can be determined by comparing sequences when aligned to maximize overlap and identity but minimize sequence gaps. In particular, sequence identity can be determined using any of a number of mathematical algorithms. A non-limiting example of a mathematical algorithm used to compare two sequences is Karlin &Altschul's algorithm (Proc.Natl.Acad.Sci.USA 1990, 87, 2264-2268), which is Karlin & Altschul. Modified by Altschul (Karlin & Altschul, Proc.Natl.Acad.Sci.USA 1993,90, 5873-5877).
Another example of a mathematical algorithm used to compare arrays is the Myers & Miller algorithm (Myers & Miller, CABIOS 1988, 4, 11-17). Such algorithms are included in the ALIGN program (version 2.0) (the ALIGN program is part of the GCG sequence alignment software package). When utilizing the ALIGN program to compare amino acid sequences, the PAM120 weight residue table, gap length penalty, 12 and gap penalty, 4 can be used. Yet another useful algorithm for identifying regions for local sequence similarity and alignment is the FASTA algorithm described by Pearson & Lipman (Pearson & Lipman, Proc. Natl.Acad.Sci.USA 1988, 85, 2444-2448). ).

In general, amino acid sequence comparisons can be achieved by aligning the amino acid sequence of a known structure polypeptide with the amino acid sequence of an unknown structure polypeptide. Then, the amino acids in the sequence are compared, and the homologous amino acid groups are divided into the same group. This method detects conserved regions of the polypeptide and reveals amino acid insertions and deletions. Homology between amino acid sequences can be determined using commercially available algorithms (see also the description of homology above). In addition to those specifically described herein, BLAST, gap-added BLAST, BLASRN, BLASTP, and PSI-BLAST programs (provided by the National Center for Biotechnology Information) are also mentioned. These programs are widely used in the art for this purpose and can align homologous regions of two amino acid sequences.
In all of these sets of search programs, the gap addition alignment routine is essential for the database search itself. Gap addition can be stopped if desired. The initial penalty (Q) for a gap of length 1 is Q = 1 for protein and BLASTP and Q = 10 for BLASTN, but may be changed to any integer. The initial setting one-residue penalty (R) for extending the gap is R = 2 for proteins and BLASTP and R = 10 for BLASTN, but may be changed to any integer. Any combination of values for Q and R can be used to align the sequences to maximize overlap and identity but minimize sequence gaps. The default amino acid comparison matrix is BLOSUM62, but other amino acid comparison matrices (eg PAM) are also available.

The terms "protein", "polypeptide" and "polypeptide fragment" are used interchangeably herein to refer to an amino acid residue polymer of any length.
As used herein, the term "polynucleotide" refers to a polymeric form of any length of nucleotide, said said to include deoxyribonucleotides or ribonucleotides.
As used herein, the term "vector" refers to recombinant DNA or RNA plasmids or viruses, which include heterologous polynucleotides to be delivered, for example, in vivo to target cells. The heterologous polynucleotide can contain the sequence of interest for therapeutic purposes and may optionally be in the form of an expression cassette. As used herein, the "vector" need not be capable of replicating in the final target cell or animal of interest.
As used herein, the term "recombinant" means a polynucleotide of semi-synthetic or synthetic origin, which is linked to another polynucleotide in an organization that is not naturally present or found in nature. ..
As used herein, the term "heterogeneous" means that an entity is derived from an entity that is genetically distinct from the rest of the entity being compared. For example, genetic engineering can place a polynucleotide within a plasmid or vector from a different source, thus the polynucleotide is a heterologous polynucleotide. A promoter taken from its original coding sequence and ligated to act on a coding sequence other than that original sequence is therefore a heterologous promoter.
Polynucleotides used in accordance with the present invention are, for example, additional coding sequences within the same transcription unit, regulatory elements (eg, promoters), ribosome binding sites, transcription termination factors, polyadenylation sites, additions under the control of the same or different promoters. Transcriptional units, additional sequences such as sequences that allow cloning, expression, homologous recombination of host cells, and any constructs that can be desired to provide embodiments of the invention can be included. ..

In certain features, the present disclosure provides a method of treating a subject mammal suffering from or at risk of osteoarthritis, wherein the method encodes a bone-protective or regenerative polypeptide. And optionally comprising administering to said subject mammal a therapeutically effective amount of adeno-associated virus (AAV) comprising a nucleic acid sequence ligated to act on a promoter, wherein the polypeptide is said to be said. It is expressed in vivo in an amount effective for alleviating or preventing the symptoms of OA in the target mammal. In some embodiments, administration is by the intra-articular route.
In some embodiments, the polypeptide is hyaluronic acid synthase (HAS) (including HAS2), lubricin, interleukin-1 receptor (IL-1R) antagonist, insulin-like growth factor 1 (IGF-1), fiber. Blast growth factor 2 (FGF-2), transforming growth factor beta 1 (TGFβ1), bone-forming protein 7 (BMP7), glucosamine-fluctose-6-phosphate aminotransferase (GFAT), interleukin 10 (IL-10) , Hemoxygenase-1 (HO-1), the biologically active incisal form described above, or the combination described above. In certain embodiments, the target mammal may be a human, dog or cat. In a specific embodiment, the target animal is a dog.
In some embodiments, the subject mammal suffers from or is at risk of developing chronic osteoarthritis.

In another embodiment, the polypeptide is canine HAS2 or canine lubricin. In some embodiments, the nucleic acid sequence encoding the HAS2 polypeptide has a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 3, or the nucleic acid sequence encoding the rubricin polypeptide is a sequence. It has a sequence that is at least 90% identical to the sequence shown in number: 6.
In some embodiments, the HAS2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the HAS2 polypeptide exhibits at least 90% identity with the sequence set forth in SEQ ID NO: 2, fragments, variants and homologues thereof, each of which exhibits in vivo HAS activity in a subject animal. ) Has an amino acid sequence selected from. "HAS activity" means the production of biologically active hyaluronic acid.
In some embodiments, the AAV vector comprises the following elements from 5'to 3': 5'AAV ITR, stuffer, CBA, intron (IN), cHAS 2-codon optimized cDNA, polyadenylation signal (pA). , And 3'AAV ITR.
In some embodiments, the rubricin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 7. In another embodiment, the rubrisin polypeptide is a polypeptide having at least 90% identity with the sequence set forth in SEQ ID NO: 7, fragments, variants and homologues thereof (the above, each exhibiting in vivo rubrisin activity in a subject animal). Has an amino acid sequence selected from. "Lubricin activity" means providing lubrication in substantially the same manner and to substantially the same degree as endogenously produced lubricin. Such lubricity activity can be measured according to techniques known in the art (see, eg, Swan, DA et al. Biochem J.1985 Jan 1; 225 (1): 195-201).
In some embodiments, the promoters are CMV IE promoter, RSV promoter, HSV-1 TK promoter, SV40 early promoter, SV40 late promoter, adenovirus major late promoter, phosphoglycerate kinase gene promoter, metallothioneine gene promoter, α- 1 Antitrypsin gene promoter, albumin gene promoter, collagenase gene promoter, elastase I gene promoter, CBA promoter, β-actin gene promoter, β-globin gene promoter, γ-globin gene promoter, α-fetoprotein gene promoter, and muscle creatin kinase (CK) You can choose from the group consisting of gene promoters.
In yet another embodiment, the AAV comprises an AAV2 or AAV5 capsid.

In another feature, the present disclosure provides a method of increasing hyaluronic acid production in both chondrocytes and / or synovial cells of a mammal (eg, human or dog animal). In certain embodiments, the method expresses a step of administering a recombinant AAV (“rAAV”) containing an rAAV vector genome (including a nucleic acid encoding HAS2) to a mammal (eg, human or dog), the HAS2 enzyme. The step may then allow sufficient time to catalyze the production of additional hyaluronic acid, thereby increasing hyaluronic acid levels in the mammal.
The present disclosure also provides a method of increasing the production of rubricin polypeptide in both mammalian (eg, human or dog) chondrocytes and / or synovial cells. In certain embodiments, the method comprises administering rAAV containing the rAAV vector genome (including a nucleic acid encoding lubricin) to a mammal (eg, a human or dog), sufficient time for lubricin to be expressed. Tolerate and thereby increase canine rubricin levels can be included.
In certain embodiments, HAS2 is produced in sufficient amount after administration of rAAV containing a nucleic acid encoding HAS2 to treat or prevent the symptoms of OA in a mammal (eg, human or dog).
In another embodiment, lubricin is produced in sufficient amount after administration of rAAV containing the nucleic acid encoding lubricin to treat or prevent the symptoms of OA in mammals (eg, humans or dogs).
In certain embodiments, HA levels are restored to the levels found in healthy mammals (eg, humans or dogs). Those skilled in the art can examine various references to find out what HA levels are found in healthy animals (eg Smith, GN et al. Arthritis Rheum. 1998; 41: 976-985; Balazs E. et al. Disorders of the Knee. Philadelphia: JB Lippincott; 1982.pp.61-74).
In another embodiment, lubricin levels are restored to the levels found in healthy mammals (eg, humans or dogs).

In another feature, the disclosure provides a method of treating dogs with or at risk of developing OA, wherein the method encodes a HAS2 or lubricin polypeptide operably linked to a promoter. Including a step of administering to the dog a therapeutically effective amount of an AAV vector containing the nucleic acid sequence to be used. In another embodiment, the disclosure provides a method of treating a human who has or is at risk of developing OA, wherein the method comprises HAS2 or rubricin polypeptide operably linked to a promoter. It comprises the step of administering to said human a therapeutically effective amount of an AAV vector containing the encoding nucleic acid sequence.
In some embodiments, the nucleic acid sequence encoding the HAS2 polypeptide has at least 90% identity with the nucleic acid sequence set forth in SEQ ID NO: 3, or the nucleic acid sequence encoding the rubricin polypeptide is SEQ ID NO: :. It has at least 90% identity with the nucleic acid sequence shown in 6.
In some embodiments, the AAV encodes a HAS2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, or comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 2. .. In some embodiments, the AAV encodes a rubricin polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7, or comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 7. ..
In any embodiment, the promoters are CMV IE promoter, RSV promoter, HSV-1 TK promoter, SV40 early promoter, SV40 late promoter, adenovirus major late promoter, phosphoglycerate kinase gene promoter, metallothioneine gene promoter, α-1. Antitrypsin gene promoter, albumin gene promoter, collagenase gene promoter, elastase I gene promoter, β-actin gene promoter, CBA promoter, β-globin gene promoter, γ-globin gene promoter, α-fetoprotein gene promoter, and muscle creatine kinase gene You can choose from promoters.

In another feature, the present disclosure administers to said dog a therapeutically effective amount of rAAV comprising an rAAV vector genome comprising a nucleic acid sequence encoding HAS2 or rubricin polypeptide ligated to actuate on a promoter. Provided are methods, including steps, to prevent the development of OA in target mammals at risk. In certain embodiments, the nucleic acid sequence encoding the HAS2 polypeptide has at least 90% identity with the nucleic acid sequence set forth in SEQ ID NO: 3, or the nucleic acid sequence encoding the rubricin polypeptide is in SEQ ID NO: 6. Has at least 90% identity with the nucleic acid sequence shown. In another embodiment, the nucleic acid encodes a HAS2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, or the nucleic acid encodes a rubricin polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the promoters are CMV IE promoter, RSV promoter, HSV-1 TK promoter, SV40 early promoter, SV40 late promoter, adenovirus major late promoter, phosphoglycerate kinase gene promoter, metallothioneine gene promoter, α-1 anti. A group consisting of a trypsin gene promoter, an albumin gene promoter, a collagenase gene promoter, an elastase I gene promoter, a β-actin gene promoter, a β-globin gene promoter, a γ-globin gene promoter, an α-fetoprotein gene promoter, and a muscle creatin kinase gene promoter. You can choose from. In some embodiments, the rAAV vector comprises CBA-cHAS2co-BGH. In another embodiment, the rAAV vector comprises pITR / minCBA-HIb-cLub1co-BGH.

In another embodiment, the disclosure provides a recombinant plasmid vector comprising a nucleic acid sequence encoding a canine HAS2 or rubricin polypeptide operably linked to a promoter. In some embodiments, the nucleic acid sequence encoding the HAS2 polypeptide has at least 90% identity with the nucleic acid sequence set forth in SEQ ID NO: 3, or the nucleic acid sequence encoding the rubricin polypeptide is SEQ ID NO: :. It has at least 90% identity with the nucleic acid sequence shown in 6. In some embodiments, the nucleic acid encodes a HAS2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, or the nucleic acid encodes a rubricin polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7.
In certain features, the present disclosure provides a pharmaceutical composition, wherein the pharmaceutical composition is a recombinant viral vector that encodes and expresses HAS or rubricin in vivo in a mammalian host, and optionally one or more pharmaceuticals. Includes acceptable carriers, excipients or vehicles.
In another embodiment, the disclosure provides a method of treating a subject mammal suffering from or at risk of developing osteoarthritis, wherein the method is the treatment of the pharmaceutical composition detailed above. Including a step of intra-articular administration of a particularly effective amount to the target mammal. In certain embodiments, the target animal is a human or dog.
In another feature, the disclosure provides adeno-associated virus (AAV) -based biological delivery and expression systems used in the treatment or prevention of OA in human or mammalian joints. In some embodiments, the method is accomplished by long-term gene expression of human or mammalian HAS2 or rubricin in synovial cells and / or chondrocytes followed by delivery of rAAV, wherein the rAAV is human or mammalian. Contains a nucleic acid sequence encoding HAS2 or rubricin, left and right AAV reverse terminal repeats (LITR and RITR), AAV packaging signals, and optionally non-viral non-coding stuffer nucleic acid sequences. In some embodiments, expression of the human or mammalian HAS2 or rubricin gene in synovial and / or chondrocytes is regulated by an inflammation-inducible promoter. The promoter is located upstream of the reading frame of the nucleic acid sequence encoding HAS2 or rubricin in humans or mammals and is specifically activated by increasing levels of immunostimulatory substances.

In some embodiments, the inflammation-inducing promoter is selected from: NF-KB promoter, interleukin 6 (IL-6) promoter, interleukin-1 (IL-1) promoter, tumor necrosis factor (TNF). Promoter, cyclooxygenase 2 (COX-2) promoter, complement factor 3 (C3) promoter, serum amyloid A3 (SAA3) promoter, macrophage inflammatory protein-1a (MIP-1a) promoter and hybrid constructs described above. In some embodiments, the rAAV vector genome comprises the nucleic acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 6, or the biologically effective variant described above. In some embodiments, the AAV system comprises a nucleic acid encoding a marker gene that allows monitoring of the vector genome in synovial and chondrocytes. In some embodiments, the vector comprises the nucleic acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 6, or the conserved sequence described above encoding the same amino acid. In some embodiments, the rAAV vector genome comprises a nucleic acid encoding the HAS2 polypeptide set forth in SEQ ID NO: 2 or the rubricin polypeptide set forth in SEQ ID NO: 7. The rAAV vector genome contains a nucleic acid molecule having at least 80% or 90% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 3. In another embodiment, the rAAV vector genome comprises a nucleic acid molecule having at least 80% or 90% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 6.
In some embodiments of the AAV system, the system is a nucleic acid sequence encoding human or mammalian HAS2 or rubricin, left and right AAV reverse terminal repeats (LITR and R ITR), packaging signals, and optionally. Expression of the HAS2 or rubricin gene in humans or mammals within synovial and / or chondrocytes contains a non-viral, non-coding stuffer nucleic acid sequence for the treatment or prevention of osteoarthritis (OA). It is regulated by an inflammation-inducing promoter and specifically activated by increasing levels of immunostimulatory substances.

Viral Particles and Methods of Producing Viral Particles : Further provided herein are viral particles containing a nucleic acid encoding HAS2 or rubricin. Viral vectors can be used to deliver nucleic acids encoding HAS2 or rubricin to express proteins in target cells at specific locations (eg, joints). Many viral species are known and many have been studied for the purpose of delivering nucleic acids to target cells. Exogenous nucleic acids can be inserted into vectors (eg, adeno-associated virus (AAV)).
In some embodiments, the viral particle is a recombination comprising a nucleic acid comprising one or two AAV ITRs and a sequence encoding HAS2 or lubricin described herein flanked by one or two ITRs. AAV particles. Nucleic acid is encapsulated within AAV particles. AAV particles also contain capsid proteins. In some embodiments, the nucleic acid is a component operably linked in the direction of transcription, a control sequence (including transcription initiation and termination sequences), and a protein coding sequence of interest (eg, a nucleic acid encoding a fusion protein). including. These components are flanked at the 5'and 3'ends by a functional AAV ITR sequence. “Functional AAV ITR” means that the ITR functions as intended for the rescue, replication and packaging of AAV virions (see: Davidson et al., PNAS, 2000, 97 (7) 3428-32; Passini et al., J.Virol., 2003, 77 (12): 7034-40; and Pechan et al., Gene Ther., 2009, 16: 10-16 Also by reference in its entirety is included herein)). To implement some of the features of the invention, the recombinant vector comprises at least all of the sequences of AAV essential for encapsulation and physical structure for infection by rAAV. The AAV ITRs used in the vectors of the invention need not have wild-type nucleotide sequences (eg, those described by Kotin (Kotin, Hum.Gene Ther., 1994, 5: 793-801)) and are of nucleotides. It may be modified by insertion, deletion or substitution, or it may be derived from any of several AAV serotypes. To date, over 40 AAV serotypes have been known, and new and existing serotype variants continue to be identified. See, for example: Gao et al., PNAS, 2002, 99 (18): 11854-6; Gao et al., PNAS, 2003, 100 (10): 6081-6; and Bossis et al., J. .Virol., 2003, 77 (12): 6799-810. The use of any AAV serotype is considered to be within the scope of the present invention. In some embodiments, the rAAV vector is in AAV serotypes including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, and AAVrh.10. Derived vector. In some embodiments, the nucleic acid of AAV comprises an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, or AAVrh.10. In yet another embodiment, the rAAV particles include a capsid protein of AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, or AAVrh.10.

Various AAV serotypes are used to optimize transduction of specific target cells or to target specific cell types within specific targets (eg, joints). The rAAV particles can contain viral proteins and nucleic acids of the same serotype or mixed serotype. For example, rAAV particles can contain an AAV2 capsid protein and at least one AAV2 ITR, or said can include an AAV2 capsid protein and at least one AAV5 ITR. In another example, rAAV particles can contain an AAV5 capsid protein and at least one AAV2 ITR. As used herein, any combination of AAV serotypes for the production of rAAV particles is provided as if each combination was explicitly described herein.
rAAV particles can be produced using methods known in the art. See, for example, US Pat. Nos. 6,566,118, 6,989,264, 6,995,006. In the practice of the present invention, host cells that produce rAAV particles include mammalian cells, insect cells, plant cells, microorganisms and yeast. The host cell can also be a packaging cell, in which the AAV rep and cap genes can be stably maintained in the host cell or producer cell (where the AAV vector genome is stably maintained). Exemplary packaging and producer cells are derived from 293, A549 or HeLa cells. AAV vectors are purified and formulated using standard techniques known in the art.

Some features provide a method of producing any rAAV particles disclosed herein, said method comprising: culturing host cells under conditions in which rAAV particles are produced: In the host cell, (i) one or more AAV packaging genes (each of which encodes an AAV replicating or encapsulating protein), (ii) a book flanked by at least one AAV ITR. An rAAV provector containing a nucleic acid encoding any fusion protein disclosed herein, and (iii) including AAV helper function); and (b) the step of recovering rAAV particles produced by the host cell. In some embodiments, the at least one AAV ITR is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, and AAVrh.10 ITR. In some embodiments, the encapsulated protein is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, and AAVrh.10 capsid proteins. In yet another embodiment, the rAAV particles are purified. As used herein, the term "purified" lacks at least some of the other components that may also be present where the rAAV particles are naturally present or where the rAAV particles were originally prepared. Includes particle preparation. Thus, for example, isolated rAAV particles can be prepared using purification techniques that concentrate the particles from a mixture of sources (eg, culture lysate or production culture supernatant). Concentration can be measured by a variety of methods, such as the proportion of DNase resistant particles (DRPs) present in solution or by infectivity. The above can also be measured in comparison to a second potential interfering substance (eg, a contaminant) present in the source mixture. The contaminating substance includes a contaminating substance in production culture or a contaminating substance at the time of processing (including helper virus, medium component, etc.).
Furthermore, what is provided herein is a pharmaceutical composition comprising rAAV particles containing a nucleic acid encoding HAS2 or lubricin of the invention and a pharmaceutically acceptable carrier. The pharmaceutical composition may be suitable for the various modes of administration described herein, including, for example, systemic or topical administration. The pharmaceutical composition of rAAV containing the nucleic acid encoding HAS2 or lubricin described herein can be systemically, for example, by intravenous injection, by catheter (see US Pat. No. 5,328,470), or by stereotactic injection (Chen et al). ., 1994, PNAS, 91: 3054-3057) Can be introduced. In some embodiments, the compositions comprising rAAV and pharmaceutically acceptable carriers described herein are suitable for administration to humans. Such pharmaceutically acceptable carriers can be sterile liquids such as water and oils (including oils of petroleum, animal, plant or synthetic origin) such as peanut oil, soybean oil, mineral oil and the like. Aqueous saline and dextrose water, polyethylene glycol (PEG) and glycerol solutions can also be utilized as liquid carriers, especially for injectable solutions. Pharmaceutical compositions can further include additional ingredients such as preservatives, buffers, tonics, antioxidants and stabilizers, nonionic wetting or clarifying agents, thickeners and the like. The pharmaceutical compositions described herein can be packaged in single-unit or multi-unit dosage forms. The composition is generally formulated as a sterile, substantially isotonic solution.

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Yoshida M et al.Expression analysis of three isoforms of hyaluronan synthase and hyaluronidase in the synovium of knees in osteoarthritis and rheumatoid arthritis by quantitative real-time reverse transcriptase polymerase chain reaction.Arthritis Research Therapy 2004, 6: R514-R520

Construction and evaluation of HAS2 AAV vectors
Example 1a-Summary An rAAV vector containing a codon-optimized canine HAS2 cDNA and a ubiquitous promoter was generated and packaged in an AAV2 or AAV5 capsid. Large vector lots were made by triple transfection and purified by CsCl gradient. Vector yields were quantified by qPCR for bovine growth hormone (BGH) poly A site (pA). A total of 4 lots were made for AAV2 / HAS2 (3 lots of which were pooled for in vivo testing, 2x10 ¹³ DRP / total volume). For AAV5 / HAS2, 2 lots (5x10 ¹² DRP / total volume) were prepared and the consistency of production yield was tested to obtain a sufficient amount of virus.
Twenty-two healthy adult dogs (AAV2 and AAV5 capsid serotype negative) were injected with rAAV2 (1, 5 and 10x10 ¹¹ vg / joint), rAAV5 (5x10 ¹¹ vg / joint), or PBS (control) by intra-articular injection. It was administered. Histopathological analysis showed minimal synovial inflammation in the rAAV5 treated joints and no significant changes in the rAAV2 treated group. The vector genome (VG) was detected in the synovium of all rAAV-treated joints and in the majority of cartilage samples. The rAAV5 vector resulted in higher VG detection and mRNA expression in both tissues compared to rAAV2. An increasing tendency of HA levels in the synovial fluid of the treated joint was observed. In summary, the disclosure results reveal a gene transfer to canine joint tissue and an acceptable safety profile upon administration by a single intra-articular injection into a dog.

Example 1b-Method
Cloning and preparation of HA expression vector : The canine HAS2 gene (GenBank XM 539153.3 (SEQ ID NO: 1)) was codon-optimized by the GeneArt / Invitrogen algorithm for expression in dogs. This codon-optimized canine HAS2 cDNA (1656bp (SEQ ID NO: 3)) was synthesized using a flanking NheI-NsiI restriction enzyme site. The fragment was subsequently cloned into a plasmid containing the ubiquitous chicken b-actin promoter (CBA), hybrid intron and bovine growth hormone (BGH) poly A (pA). The obtained pCBA-HI-cHAS2-BGHpA plasmid was purified for expression analysis using a maxi kit (Qiagen).
In vitro expression of cHAS2 and HA production : A plasmid vector containing cHAS2 was transfected into 293 cells, and after 3 days, conditioned cultures and cell lysates were collected in 250 μL of RIPA buffer plus protease inhibitor. Cytolysis was rotated to remove cell debris, and 30 μL of cytolysis was loaded onto a 4-12% nu-PAGE gel and run in 1x Mops buffer. The protein gel was transferred to a nitrocellulose membrane and examined overnight at 4 ° C. using anti-HAS2 as a probe in 5% milk and 0.1% Tween-20 PBS-T. A donkey anti-goat secondary antibody (1: 5000 dilution) was used as the secondary antibody. Beta actin detection was used to show that an equal amount of cytolysis was loaded.
Quantification of HA levels and molecular weight in in vitro cultures : HA production by HAS2-expressing cells was assessed by transfecting 293 cells with pCBA-HI-cHAS2-BGHpA (in Optimen or complete medium). HA levels in conditioned cultures were quantified using an HA test kit (Corgenix, Inc.). The kit contains an HA-binding protein derived from agrecan. The molecular weight of HA was evaluated by running a concentrated conditioned culture medium on an agarose gel. Various HA size markers were run in parallel (Select-HA HiLadder, Hyalose, Austin, TX). Similar gels were run in parallel, followed by hyaluronidase digestion for 24 hours. Both gels were stained with All-stain.
Preparation of rAAV vector with cHAS: The cHAS2 expression cassette was cloned into an AAV ITR-containing plasmid to generate an expression cassette flanked by AAV reverse-terminal repeat (previrus plasmid pDC627), psITR / CBA-HI-cHAS2- Constructed BGHpA. 600 bp stuffer DNA (chromosome 16 P1 clone 96.4B) was added upstream of the expression cassette to create a total 4500 bp viral vector genome. To test the packaging of the plasmid containing the cHAS2 expression cassette, 293 cells were seeded at 8x10 ⁵ cells / well (6-well plate) and the next day psITR / CBA-HI-cHAS2-BGHpA or psp70 / EGFP, The pHLP-19cap2 or p5repCMVcap5 plasmid and pAdHELP were duplex-transfected (CaPO ₄ kit (Promega)). After 3 days, cells were harvested and lysate titers were measured for BGHpA copies using qPCR and primers / probes for BGHpA sequence (SEQ ID NO: 12-14). A plasmid containing BGHpA was used as a standard substance. The rAAV virus yield was expressed as the amount of DNase resistant particles (DRP) per cell. Large-scale vector generation was performed using triple transfections of psITR / CBA-HI-cHAS2-BGH, the pIM45BD rep-cap plasmid for the AAV2 vector, and pHLP19-cap5 and pAdHELP for the AAV5 vector. Vectors were purified by CsCl and titers of vector lots obtained using TaqMan analysis and BGHpA sequence primers / probes were measured (Applied Biosystems / Life Technologies).
In vitro efficacy of rAAV / cHAS2 in rabbit chondrocytes and synovial cells : The ability of the vector to transfect articular cell types (eg, primary synovial and chondrocytes) was tested in rabbit cells. Cells were infected with 1e5 DRP / cells and cultured for 3 days. Cytolysis was collected and HAS2 protein was detected by Western blotting, and HA levels were further quantified as described above. To test the effects of HA on symptomatic matrix-degrading protease production, inflammatory cytokines and cartilage structural protein production, cells were first infected with the rAAV vector followed by IL-1b stimulation 24 hours later. .. After 24 hours, both cells and culture medium were collected for mRNA analysis and HA production.
Evaluation of rAAV / cHAS2 in normal dog joints : Mongrel dogs (male and female, 8-10 kgkg kg) were used. Dogs with serum titers of 4 or less for AAV2 and / or AAV5 capsids were used in the study. The rAAV and rAAV5 vectors encoding cHAS2 were administered by the intra-articular route (AAV2: 1, 5 and 10x10 ¹¹ DRP / joint, AAV5: 5x10 ¹¹ DRP / joint). PBS was used as a negative control. Animals were observed for clinical signs (pain, swelling, injection joints) once daily for 7 days before injection, twice daily for 7 days after injection, and once daily during the subsequent study period. Swelling and other abnormalities). The animal was sacrificed four weeks later. Whole blood samples were collected -7, 1, 14 and 28 days after vector administration for white blood cell (WBC) measurement. Synovial fluid (SF) samples were collected on days -7, 1, 14 and 28 for quantification of HA levels. Synovial tissue, cartilage and liver samples were collected for DNA and RNA isolation. The cHAS2 vector genome and mRNA copy were determined by qPCR analysis using the BGHpA primer / probe set (Applied Biosystems / Life Technologies). The sections were stained with toluidine blue and scrutinized by a qualified veterinary pathologist. Cartilage was evaluated for cartilage lesion severity and proteoglycan reduction (score 0-5). No synovial thickening was observed, so the synovial lesion score was determined by the density of inflammatory cells (score 0-5).

Example 1c-Results
Codon optimization and production of HAS2 expression cassettes : Mammalian HAS2 is a highly conserved protein. For example, the human and canine amino acid sequences of HAS2 differ only in the two amino acids (99.3% identity). Similarly, there are only 3 amino acid differences between HAS2 in dogs and rabbits (99.5%). At the DNA level, the similarity between canine and human HAS2 synthase cDNA is 93.9%. Since gene expression can be improved by codon optimization, the GenBank sequence of canine HAS2 (XM 539153.3) was optimized by GeneArt / Invitrogen. This gave a nucleotide sequence with 78% similarity to the original GenBank sequence. The GC content of the optimized cDNA increased from 44.4% to 59.0%. Using this cDNA, a ubiquitous expression plasmid with a CBA promoter was prepared to enable constitutive expression of HAS2, which is different from the endogenous promoter (Fig. 1A). The CBA promoter is less affected by various pre-inflammatory and anti-inflammatory cytokines.
HAS2 expression and HA production in vitro: To test the in vitro expression of HAS2 protein, two clones (# 1 and # 2) of the CBA-HI-cHAS2-BGHpA plasmid vector were transfected into 293 cells, followed by Cytolysis was analyzed for HAS2 protein (membrane protein) by Western blotting. Cells transfected with the expression plasmid showed a band of 64 kDa, the expected size of cHAS (data not presented). We then determined whether overexpression of the HAS2 protein in 293 cells resulted in increased detection of HA in culture (both HA production and HA secretion across the cell membrane). HA levels in the medium of cells transfected with pCBA-cHAS2 were increased 6.5-fold and 9-fold, respectively, compared to non-transfected cells and CBA-EGFP-transfected cells (Fig. 1B). Therefore, the data confirmed that overexpression in cells resulted in increased HA levels in the extracellular compartment. The size of HA produced in vitro was determined on an agarose gel. The data showed the presence of high molecular weight HA in conditioned cultures obtained from cells transfected with the HAS2 expression cassette. The size of this material was greater than 1.5 megadaltons (MDa) (based on estimates by HA molecular weight markers). This substance disappeared after hyaluronidase digestion, indicating that the substance was HA (Fig. 1C).
Preparation of rAAV vector with HAS2 expression cassette : The cHAS2 co-expression cassette was subsequently cloned into a plasmid with AAV ITR. A schematic diagram of the obtained viral genome is shown in FIG. 1A. The ability to generate rAAV vectors with AAV2 and AAV5 capsids as well as HAS2 cDNA was tested in small packaging experiments (Fig. 2A), followed by large-scale vector generation. Both AAV2 and AAV5 vectors could be generated using standard triple transfection methods (Fig. 2B). The capacity of this material was tested by infection of 293 cells and analysis of production HA levels in culture. Both AAV2 and AAV5 vectors resulted in an increased dose response of HA in culture (Figures 2C, D).
Evaluation of rAAV / HAS2 vector in normal dog joints : rAAV2 and rAAV5 vectors with cHAS2 were delivered to normal dog joints by intra-articular administration and animals were evaluated for 28 days. No adverse clinical signs, weight changes (Fig. 3A), repellency or death were observed during the study. -On day 7 some animals have increased white blood cell (WBC) counts, which may be due to transport stress. The WBC numbers on days 1, 14 and 28 were generally within the normal limits. Histological assessment of PBS-, AAV-injected (left) and contralateral (non-injected) knees showed very slight proteoglycan reduction and cartilage degeneration (score range 0-0.5 (maximum score 5)). Shown. These minimal changes were typical age-appropriate spontaneous changes. Minimal synovial changes were observed in PBS- and AAV2-treated joints as well as contralateral joints (Fig. 3C). Minimal to mild synovitis (generally spreading to the joint capsule and medial collateral ligament) was observed in all left knees of males and females treated with the AAV5 vector (synovitis observed in the contralateral joint) Was not done). Therefore, overall, this procedure was well tolerated with few observed adverse effects.
Tissue samples collected from the synovium and cartilage for detection of the viral genome were analyzed (Figs. 4A, 6A). Synovial samples collected from the nearest injection site (Sample # 3) showed the presence of the vector genome in all AAV-treated joints (Fig. 4B). AAV2-treated joints roughly contained 0.01 to 2 vector genomes (VG) / cells. Interestingly, a minor dose response was observed with AAV2, despite a 10-fold difference between the low-dose and high-dose groups. Joints treated with the AAV5 vector showed higher and more consistent detection, ranging from 1 to 12 copies / cell. Low levels of VG were detected in some contralateral (non-injection) joints, which were more pronounced in the low AAV2 treatment group and more sporadic in the high AAV2 dose group and the AAV5 group (data presented). Not done).
Synovial samples (Sample # 1) collected further above the injection site were analyzed to assess AAV diffused into the joint (Fig. 4C). Joints injected with low-dose AAV2 showed more consistent VG detection. These levels are similar to those measured in synovial sample # 3. In the AAV5 treatment group, all synovial # 1 samples have consistently detectable VG (within 3x). However, these were lower than the VG levels detected in synovial membrane # 3, thus revealing position-dependent transduction.
Expression from the vector genome was analyzed by quantification of vector-derived mRNA. For synovial sample # 3, expression was detected in 2/5, 4/5, and 4/5 of the AAV2-treated low, medium, and high-dose groups, but all AAV5-treated joints had detectable mRNA copies. (Fig. 5A). Vector expression was also detected in synovial membrane # 1 for the AAV5 vector, but its levels were as low as low VG detection at this location (Fig. 5B). The detection of mRNA showed a good correlation with the detection of VG. The respective mRNA and VG DNA of the individual injection joints of synovial sample # 3 are shown as examples (Fig. 5C).

Detection of vector genomes in canine cartilage : Cartilage samples collected from the femoral condyle and tibial plateau were analyzed for detection of the viral genome (VG, Figure 6A). Vector DNA and mRNA detected in each of the individual injection joints and group averages in the femoral condyle are shown as examples (FIGS. 6B, C). This data showed that the AAV5 vector was present in cartilage in a consistent manner and showed comparable levels of vector-derived transcripts. Similar doses of the AAV2 vector (medium dose) produced similar VG levels as the AAV5 vector, but showed 100-fold lower mRNA levels. In addition, AAV2 VG copies appeared to have an inverse correlation with vector doses. The rAAV5 injection joint also showed vector detection and expression in cartilage samples collected from the tibial plateau, but not at all in the rAAV2-treated joint (Fig. 6D). All vectors resulted in minimal vector DNA detection in the contralateral (non-injection) joint.
Synovial and cartilage results are summarized in Figure 7A. For gene transfer in the synovium, the AAV5 vector yielded a vector copy approximately 10-fold higher at the location of both synovial samples compared to the AAV2 vector. Gene transfer into cartilage was 10 to 20-fold lower than gene transfer into the synovium by AAV5, while the AAV2 vector genome was observed at similar levels in both synovium and cartilage. Detection of the rAAV5 vector-derived genome and mRNA is summarized in Figure 7B, demonstrating consistent gene transfer and expression with the rAAV5 / HAS2 vector in all tissue samples tested. Applicants believe that this result is highly disappointing.
Analysis of synovial fluid HA levels : Synovial HA levels were measured on days-7 (reference line) and to determine if any changes in synovial fluid HA levels could be detected after administration of the rAAV vector. Quantified with samples collected on day 28. High levels of variation were detected between animals, so HA levels in each animal were normalized to the baseline level in each animal. The data showed that both AAV2 / high and AAV5 / medium doses increased synovial fluid HA levels on average compared to PBS-treated animals (FIGS. 8A and 8B).

Example 1d-Conclusion To provide in vivo overexpression of HA in joints, Applicants generated rAAV vectors using two capsid serotypes. Selection of AAV capsid serotypes is important. This is because any neutralizing antibody already present in the target species can neutralize the therapeutic vector and thus block gene transfer by the rAAV vector. The results disclosed herein showed that the majority of dogs analyzed had low levels of neutralizing antibodies against both AAV2 and AAV5 capsids. Therefore, Applicants tested the localization of AAV2 and AAV5 capsids directly in the target tissue (ie, canine knee joint) after intra-articular injection. Since HA expression is expected to be beneficial for both synovial and chondrocyte cells, vector genomic copies were quantified in canine synovial and chondrocyte samples, respectively. The data show that AAV2 provides highly inconsistent in vivo gene transfer into canine synovial and cartilage tissues, and shows little dose-responsive effect, for unknown reasons. Similar experiments performed on OA rabbit joints showed highly consistent rAAV2 vector genomic detection using similar vector doses (Kyostio-Moore, 2015). In contrast to the AAV2 vector, the AAV5 vector genome was detected in a consistent manner in both tissue types (n = 5 / group).
Importantly, the detection of AAV5 in cartilage samples is surprising and unexpected. This is because cartilage has been reported to be difficult to transduce due to the extensive extracellular matrix under in vivo conditions, and so far no other reports on the detection of AAV5 in cartilage of many animals after intra-articular delivery. Because it doesn't exist. In addition, the canine studies of the present disclosure produced surprisingly high levels of the rAAV5 vector in the canine synovial tissue, which is also about 2 logs higher in the synovium compared to that of cartilage and against the canine inner layer of AVV5. Shows priority. This preferred expression pattern was also unpredictable prior to this disclosure.
In addition to high levels of vector detection, mRNA analysis confirmed recombinant HAS2 expression in canine synovial and cartilage tissues, indicating that the CBA promoter has function in both tissue types. Furthermore, detection of transcripts by AAV5 in cartilage samples confirmed that chondrocytes were transduced by the vector rather than the virus hidden in the extracellular matrix of cartilage. For AAV2, similar levels of vector genomes and transcripts were also observed in the inner synovial layer. However, mRNA expression of AAV2 vectors is surprisingly nearly 100-fold lower than detection of the corresponding vector genome in cartilage samples, with some of the vector remaining outside the chondrocytes and possibly retained in the extracellular matrix. It suggests that it may have been done. These significant differences will only be understood after Applicants have performed significant extraordinary experiments.
The vector was administered to only one joint in each animal, but the vector genome was sometimes detected in the contralateral non-injection joint. This was mostly observed in synovial samples obtained from AAV2-treated joints. However, none of the animals in which the vector genome was observed in the contralateral joints had any detectable HAS2 transcript in these joints.
In summary, the data disclosed herein indicate that the AAV5 capsid provides better gene transfer for intra-articular delivery to canine joints. This is due to the low existing humoral immunity to AAV5 in the target animals and the ability to transduce into joint tissue after intra-articular injection. Intra-articular injection can provide gene transfer not only to the lining of the synovium but also to the chondrocytes of cartilage. Both tissue types will benefit from the increased HA synthesis provided by the gene delivery compositions and methods of the present disclosure. That is, the synovium will benefit from the increased ability of the synovial fluid to provide lubrication, and the cartilage will benefit from enhanced matrix binding and thus functioning as a scaffold with improved cartilage health. These results indicate that overexpression of HA by AAV-mediated HAS2 gene transfer to the diseased site reduces the pathology and pain of OA.

Construction and evaluation of lubricin AAV vector
Example 2a-Summary It was recently shown that intra-articular injection of recombinant lubricin reduced cartilage degeneration in a rat OA model (Flannery, 2006). However, lubricin administered to the joint has a very short half-life in synovial fluid and most proteins are removed within 72 hours (Vugmeyster, 2011). Therefore, repeated intra-articular injections are required, which are time consuming, labor intensive, stressful and even more expensive. In contrast to HAS2 (see Example 1), lubricin is encoded by a large cDNA and also contains multiple DNA repeats in its mucin-like domain, which makes it difficult to adapt to the rAAV vector and express high levels, respectively. To. To avoid this problem, Applicants generated shortened inulbricin cDNA and optimized a small expression cassette to enhance lubricin production. Importantly, neither the full-length inulbricin sequence nor the abbreviations of this disclosure were known prior to this disclosure.
Briefly, Applicants generated cDNA for full-length inulbricin, which was subsequently used to design a shortened codon-optimized inulbricin version (cLub1co). The latter was subsequently used to construct a variety of rubricin expression plasmids. These plasmids were characterized for rubricin mRNA and protein production after transfection of HEK293 cells. The data showed that both rubrisin mRNA and secreted rubrisin were produced from each construct. Finally, Applicants generated an rAAV vector with a cLub1 expression cassette and presented the feasibility of rAAV / cLub1 vector generation. HEK293 cells infected with this construct synthesized and secreted inulbricin.

Example 2b-Method
Cloning of Inulbricin : Since there is no canine full-length rubricin cDNA in GenBank (incomplete sequence: GenBank no.ABD38836.1), complete canine cDNA was obtained from the custom synthetic canine cartilage cDNA library. To achieve the above, overlapping fragments were made using qPCR with various primers. Subsequently, a short form of inulbricin (cLub1) was designed using full-length cDNA (SEQ ID NO: 4) (similar to the short published version of human rubricin (Flannery, 2009)). This short inulbricin contained a deletion in the sequence encoding amino acids 378-782. This shortened lubricin sequence was codon-optimized (cLub1co) and further synthesized (GeneArt / Invitrogen). The cLubco fragment (smoothed KpnI to PmeI) was cloned to the Mfe (smoothed) -PmeI site of the plasmid. The plasmid contained a CMV enhancer, chicken β-actin promoter and shortened hybrid intron (Hib) (minCBA), and bovine growth hormone (BGH) polyadenylation site. E. coli Stable II cells were transformed with the ligation reactant and grown at 30 ° C. to minimize DNA rearrangement. The resulting clones were analyzed by restriction enzyme analysis and the cloning junctions were analyzed by DNA sequencing. Additional constructs were made. The additional construct contained modifications of the two "ATG" sequences present in the 6x histidine (6xHis) and intron sequences. In vitro lubricin expression was analyzed using the expression plasmid.
Invitrogen expression analysis : HEK293 cells were transfected with the rubricin expression plasmid using Lipofectamine 2000 (Invitrogen) and the cells were grown for 72 hours. To analyze rubricin mRNA expression, cells were collected and transcript levels were measured by real-time (RT) qPCR assay. BGA pA-specific primers / probes were used in the assay (7500 Real-Time PCR System; Applied Biosystems, Foster City, CA). Cultures were collected and concentrated approximately 20 to 30 times (100 k MWCO filter, Millipore) for protein production analysis. Samples were run on a 4-12% Bis-Tris gel or a 3-8% Tris-acetic acid (NuPAGE; Thermo Fisher Scientific) SDS-PAGE gel (reduction) with MOPS or Tris-acetate buffer, respectively. Lubricin was detected by Western blotting using mouse anti-lubricin antibody (9G3, Millipore) (Ai, 2015) and goat anti-mouse-HRP (Jackson ImmunoResearch Laboratories, West Grove, PA) as a secondary antibody.
Preparation of AAV / cLub1co: The cLub1 expression cassette was cloned with an AAV reverse terminal repeat (ITR) -containing plasmid to prepare an expression cassette (previrus plasmid pDC627) flanked by AAV ITR, and psITR / minCBA-HI-cLub1co was prepared. -Builded BGHpA. To test the packaging of the plasmid containing the cLub1 expression cassette, 293 cells were seeded at 8x10 ⁵ cells / well (6 well plate) and the next day psITR / minCBA-HI-cLub1co-BGHpA, or psp70 / EGFP, The pHLP-19cap2 (AAV2) or p5repCMVcap5 (AAV5) plasmid and pAdHELP (Promega CaPO ₄ kit) were transfected to package the vector in AAV2 or AAV5 capsid. After 3 days, cells were harvested and lysates were quantified for vector yield by qPCR assay (7500 Real-Time PCR System). Standard curves of BGH pA sequence-specific primers / probes (Applied Biosystems / Life Technologies) and serially diluted linearized plasmid DNA (including BGH pA) were used. The rAAV virus yield was expressed as the amount of DNase-resistant particles (DRP) per cell (Clark, 1999).
Experimental scale vector generation was performed using triple transfections of psITR / minCBA-HI-cLub1co-BGH, pHLP19-cap5 (for AAV5 vector) and pAdHELP. Vectors were purified by CsCl gradient and yields were quantified as described above (University of Massachusetts Medical School, Worcester, MA).

Example 2c-Results
Preparation of short inulbricin: The inulbricin sequence present in GenBank lacks a large portion of exon 6 (encoding 857 amino acids), so the full-length inulbricin cDNA (4017 bp (without stop codon), SEQ ID NO:: 4) was prepared. The full-length inulbricin cDNA encodes a protein containing a total of 1339 amino acids (SEQ ID NO: 5, Figure 9) (slightly smaller than the human sequence of 1404 amino acids). At the amino acid level, this inulbricin has 79% identity with the sequence of human rubrisin (SEQ ID NO: 11, FIG. 16).
Full-length inulbricin was too large to fit the rAAV vector due to packaging limitations, so a shortened version of inulbricin was made. This short inulbricin version (“Lub1”) was created by deleting the sequence encoding amino acids 378 to 782 in the mucin-like domain and is a 2949 bp long cDNA encoding 983 amino acids (SEQ ID NO: 7). (SEQ ID NO: 6) was obtained. Despite a large deletion of the mucin-like domain, nearly 10 KEPAPTT-like peptide repeats remained. Importantly, none of these are identical to the canonical human repeat sequence, but those skilled in the art (even if they are identical) believe that delivery of this shortened canine Lub1 may be effective in treating OA. You couldn't have expected it. These repeats are considered important for lubrication properties as they are potential O-linked oligosaccharide binding sites. This short lubricin codon optimization (Lub1co, SEQ ID NO: 6) increased the GC content from 44% to 60% and had 74% nucleotide similarity to the original canine DNA sequence. Subsequently, this short canine cDNA was used to generate a plasmid expression cassette carrying the minCBA promoter, cLub1co and BGHpA (Fig. 10). Expression plasmids with 6xHis-tags and alterations to the putative ATG nucleotide sequence within the intron region (minimizing false translation initiation sites) were also generated.
Expression analysis of inulbricin : Expression of cLub1co, the minimal CBA promoter (minCBA-cLub1co) plasmid, was confirmed in vitro by showing increased mRNA levels in transfected 293 cells (Fig. 11A). The activity of the minCBA-cLub1co construct with a short intron was about 3-fold lower than when the full-length CBA-HI construct (CBA-cLub1co) was used. Transcription was rarely observed with plasmids containing full-length lubricin and non-codon-optimized constructs (CBH-cLubr). Transcript analysis was also performed on expression cassettes with various modifications (FIGS. 10, 11B). Expression of minCBA-Lub1co was comparable to EGFP and C-terminal 6xHis-tag containing constructs. Deletions of the two putative ATG codons present in the hybrid intron appeared to enhance expression levels by approximately 2-fold. Another morphological change observed in Lubico-transfected cells also suggested Lub1 expression. This is because those changes were not present in non-transfected cells or EGFP plasmid-transfected cells (data not presented).
The production of canine Lub1 protein from various expression plasmids was tested by Western blot with an antibody against lubricin and showed 250-380 kDa protein in concentrated culture medium (Fig. 12A). The expected size based on 1339 amino acids is approximately 160 kDa, but the larger and broader pattern signals are probably due to glycosylation. Little detection was observed in non-transfected cells or EGFP plasmid-transfected cells. In addition, the ΔATG modification appeared to increase rubricin detection as well as observations of elevated transcript levels derived from this construct. Protein expression was also confirmed from the previral AAV plasmid, indicating detection of comparable proteins (Fig. 12B). In summary, these results demonstrate that plasmids containing the inulbricin expression cassette express and secrete glycosylated rubricin proteins.
Preparation of rAAV Vectors Containing Inulbricin Expression Cassettes : Having confirmed inulbricin expression in the plasmid vector, we then tested in small package experiments whether the expression cassettes could be packaged in AAV2 and AAV5 capsid serotypes (Figure). 13A, B). The data showed packaging efficacy of inulbricin similar to that observed with the EGFP expression vector for both AAV2 and AAV5 capsids. The addition of the 6xHis-tag did not change the rAAV vector yield. About 5-fold lower levels of packaging were measured with AAV2 vectors containing canine HAS2 expression cassettes. Subsequently, experimental-scale AAV5 / minCBA-cLub1 generation was performed to evaluate scale-up vector generation. Vector yields were comparable to standard AAV2 and AAV5 vector yields containing EGFP as a transgene (data not presented). Subsequently, the AAV5 vector was tested in vitro for lubricin production and secretion in HEK293 cells. Analysis of conditioned cultures by Western blotting showed dose-dependent detection of inulbricin (Fig. 14). In summary, the data showed that a shortened version of inulbricin could be used to generate the rAAV vector, and that cells infected with this vector could mediate lubricin synthesis and secretion into the medium.

Example 2d-Conclusion As shown above, rubricin as a transgene presents many problems for rAAV production. First, the size of the rubricin cDNA containing the required expression elements exceeded the packaging capacity of rAAV, thus requiring shorter cDNA. Interestingly, the complete KEPAPTT repeat is absent in the canine sequence compared to the human rubrisin amino acid sequence of the mucin-like domain (Fig. 16). For the production of recombinant rAAV vectors, repeating any DNA sequence can pose a challenge. This is because repeat sequences can reduce the stability and integrity of the viral genome by causing DNA deletions and rearrangements during virus production. However, given that comparable vector yields were obtained when compared to the standard EGFP reporter vector, the disclosed (and surprising) results show that the production of rAAV virus containing and expressing a novel inulbricin sequence is possible. Shown that it is feasible. Therefore, this is the first report to reveal a single rAAV vector strategy for lubricin gene delivery.

In vivo efficacy study of AAV-HAS2 in medial meniscus ligament free (MMR) model The purpose of this study is to evaluate the efficacy of HA synthase 2 gene therapy using gross observation and histology in a canine OA posterior knee joint model. Met. Twelve intact research breeding mongrel males (Foxhound phenotype, approximately 20-23 kg) were anesthetized and arthroscopically performed medial meniscus ligament release (MMR) of the right posterior knee joint (-14). Day).
Phosphate buffered saline (PBS control) or canine hyaluronic acid synthase 2 (cHAS-2) -carrying recombinant AAV5 DNase-resistant particles (DRP) (5x10 ¹¹ ) were intra-articularly administered on day 0 (n = 6 animals). /group).
Plasma was collected from all dogs on days 0 and 182 for biomarkers of arthritis. Right and left synovial fluids were collected from all PBS control and cHAS-2 treatment groups on days 0, 56, 112 and 182 for analysis of HA levels.
On day 182, the dog was euthanized, and the cartilage defect (indicated by ink staining) induced by the release of the posterior knee joint ligament was measured. Joint tissue was collected for physical examination.
Gross and histological data were analyzed by Kruskal-Wallis using GraphPad Prism 6 statistical software.
Total HA levels in synovial fluid were measured and showed no treatment-related differences in total HA levels (Fig. 17).
Synovial and cartilage samples were collected from treated joints on day 182 and analyzed for detection of the viral genome (Fig. 18A). Vector-derived DNA and mRNA were detected in each synovial sample (Fig. 18A) and most cartilage samples (Fig. 18B) of individual rAAV5 / cHAS-2 injection joints. The data are summarized in Figure 18C, showing the mean vector genome and mRNA in both tissue samples.
There was no evidence of local or systemic toxicity closely associated with intra-articular administration of HA synthase-2 gene therapy. There was consistent cartilage structure conservation with cHAS-2 treatment compared to PBS treatment. Reductions in lesion size and depth on both the medial femoral condyle and the medial tibial plateau joint surface were more pronounced in 4 of 6 rAAV5 / cHAS2-treated dogs.
In FIG. 19, the histopathological scores based on Cook et al. (Cook et al., 2001) are shown in the lower left corners of the medial femoral condyle and medial tibial cartilage images (2x), respectively. Canine 994731 / PBS had widespread erosions towards the median strip and showed significant proteoglycan disappearance on both cartilage surfaces. No cartilage protective effect was observed. Dog 993107 (treated with rAAV5 / cHAS2) had a shallow lesion in the superficial zone of the femoral cartilage, but overall there was good protection of the cartilage residue and there was little loss of proteoglycan. The tibial plateau lesion was deeper towards the median strip with a mild proteoglycan deletion. Since the underlying cartilage is relatively normal, there was a cartilage protective effect in the femoral condyle. Dog 992879 treated with rAAV5 / cHAS2 showed some proteoglycan disappearance in the femoral cartilage, but the overall morphology was preserved. The tibial plateau had well-defined nest-like erosions, but most of the cartilage was preserved. Therefore, the lesions were small and mild in severity, demonstrating some cartilage protection.
In particular, one of the rAAV5-treated animals for which the vector could not be detected in the cartilage sample also had the largest tibial plateau lesion area (dog 993107, Figure 19). Conversely, one rAAV5-treated animal (dog 992879) with vectors detected in both synovium and cartilage (but no mRNA in cartilage) had the best cartilage structure.
Therefore, the presence of the rAAV5-HAS2 vector is closely related to the best cartilage structure, and the absence of the vector is closely related to the largest tibial plateau lesion area. Therefore, despite variations in vector / mRNA detection, the rAAV5 vector expressing HAS2 appears to have produced the desired clinical results.
Taken together, these results revealed consistent rAA5-mediated gene transfer into the synovium and cartilage of canine OA joints, confirming sustained vector-derived expression for at least 6 months. Histological analysis showed that the majority of cHAS-2 treated joints reduced cartilage lesions and slowed disease progression, while little difference was observed in total synovial fluid HA levels. The latter may indicate that local HA expression in cartilage and synovium has some disease-modifying properties without increasing total synovial HA levels. Alternatively, changes in molecular weight in synthesized HA that cannot be detected by total HA level measurements may also contribute to the beneficial effects of rAAV5 / cHAS-2.

References
Sanderson RO et al. Systematic review of the management of canine osteoarthritis.Veterinary Record (2009) 164, 418-424
McIlwraith CW.Frank Milne Lecture: from arthroscopy to gene therapy: 30 years of looking in joints. Am Assoc Equine Pract 2005; 51: 65-113.
Cook et al.The OARSI histopathology initiative --recommendations for histological assessments of osteoarthritis in the dog. Osteoarthritis Cartilage, 2010; 18 suppl 3: S66-79.

The present invention is further represented by the following numbered items:
1. 1. A method of treating a target mammal suffering from osteoarthritis (OA), comprising intra-articular administration of a therapeutically effective amount of recombinant adeno-associated virus (rAAV) to the target mammal. , Containing a nucleic acid encoding a bone-protective or regenerative polypeptide linked so that the recombinant adeno-associated virus can act on a promoter, in order for the polypeptide to alleviate the symptoms of OA in the subject mammal. The method described above, which is expressed in vivo in an effective amount.
2. Polypeptides are hyaluronic acid synthase (HAS), lubricin, interleukin-1 receptor (IL-1R) antagonist, insulin-like growth factor 1 (IGF-1), fibroblast growth factor 2 (FGF-2), traits. Conversion growth factor beta 1 (TGFβ1), bone morphogenetic protein 7 (BMP7), glucosamine-fluctose-6-phosphate aminotransferase (GFAT), interleukin 10 (IL-10), hemoxygenase-1 (HO-1), supra. The method of item 1, wherein is a biologically active incisal form, or a combination of the above.
3. 3. The method of item 1 or 2, wherein the polypeptide is a HAS2 polypeptide.
Four. The method according to any one of items 1-3, wherein the target mammal is a human, dog or cat.
Five. The method according to any of items 1-4, wherein the target mammal is a dog.
6. 5. The method of item 5, wherein the polypeptide is canine HAS2.
7. 7. Item 5 or 6 wherein the HAS2 polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 2, or said fragment, variant or homologue that exhibits HAS2 in vivo activity in the subject animal. The method described in.
8. The method of any of items 5-7, wherein the HAS2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2.
9. The method of any of items 5-8, wherein the nucleic acid encoding the HAS2 polypeptide has a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 3.
Ten. From 5'to 3', rAAV has the following elements: 5'AAV reverse terminal repeat (ITR), stuffer nucleic acid, promoter, intron (IN), cHAS2 codon-optimized cDNA, polyadenylation signal (pA), and The method according to any of items 5-9, comprising the rAAV vector genome comprising 3'AAV ITR.
11. 11. 10. The method of item 10, wherein the promoter is a chicken beta-actin (CBA) promoter.
12. The method of item 1 or 2, wherein the polypeptide is a rubricin polypeptide.
13. Item 12, wherein the rubrisin polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 7, or the fragment, variant or homologue that exhibits in vivo rubrisin activity in the subject animal. The method described.
14. 13. The method of item 13, wherein the rubricin polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 7.
15. 15. 13. The method of item 13 or 14, wherein the nucleic acid encoding the rubricin polypeptide has a nucleotide sequence that is at least 90% identical to the nucleotide set forth in SEQ ID NO: 6.
16. The method according to any of items 13-14, wherein rAAV comprises the rAAV vector genome encoded by the plasmid pITR / minCBA-HI-cLub1co-BGH.
17. 17. The promoters are CMV IE promoter, RSV promoter, HSV-1 TK promoter, SV40 early promoter, SV40 late promoter, adenovirus major late promoter, phosphoglycerate kinase gene promoter, metallothioneine gene promoter, α-1 antitrypsin gene promoter, albumin. Consists of gene promoter, collagenase gene promoter, elastase I gene promoter, β-actin gene promoter, CBA promoter, β-globin gene promoter, γ-globin gene promoter, α-fetoprotein gene promoter, and muscle creatin kinase (CK) gene promoter The method according to any of items 1-9 or 12-16, selected from the group.
18. The method of item 1, wherein the AAV comprises an AAV2 or AAV5 capsid.
19. A method of increasing hyaluronic acid production in canine chondrocytes and / or synovial cells, comprising administering rAAV to the canine, wherein the rAAV is ligated to actuate on a promoter HAS2. It contains an rAAV vector genome containing the nucleic acid encoding the enzyme, and after administration, the HAS2 enzyme is expressed to catalyze the production of additional hyaluronic acid, thereby increasing the level of hyaluronic acid (HA) in the dog. , Said method.
20. 19. The method of item 19, wherein HAS2 is produced in an amount sufficient to treat the symptoms of canine OA.
twenty one. The method of item 20, wherein the HA level is restored to the level found in healthy dogs.
twenty two. A method of treating a dog suffering from OA, comprising administering to the dog a therapeutically effective amount of rAAV, wherein the rAAV encodes HAS2 ligated to actuate on a promoter. The method described above comprising an AAV vector genome comprising a nucleic acid.
twenty three. A method of treating a human suffering from OA, comprising administering to the human a therapeutically effective amount of rAAV, wherein the rAAV encodes HAS2 ligated to actuate on a promoter. The method comprising an AAV vector comprising nucleic acid.
twenty four. An item encoding HAS2 in which the nucleic acid encoding HAS2 has at least 90% identity with the nucleotide sequence set forth in SEQ ID NO: 3 or has at least 90% identity with the amino acid sequence of SEQ ID NO: 2. The method described in any of 19-23.
twenty five. The method of any of items 19-23, wherein HAS2 has the amino acid sequence set forth in SEQ ID NO: 2.
26. A method of increasing the production of rubricin in canine chondrocytes and / or synovial cells, comprising administering rAAV to the dog, wherein the rAAV is ligated to actuate a promoter. Said method comprising an rAAV vector genome comprising a nucleic acid to be administered, wherein the rubricin is expressed after administration, thereby increasing the level of rubricin in the dog.
27. 26. The method of item 26, wherein lubricin is produced in an amount sufficient to treat the symptoms of canine OA.
28. The method of item 26, wherein the rubricin levels are restored to the levels found in healthy dogs.
29. A method of treating a dog suffering from OA, comprising the step of administering to the dog a therapeutically effective amount of rAAV, wherein the rAAV encodes a promoter-linked rubricin. The method described above comprising an rAAV vector genome comprising a nucleic acid.
30. A method of treating a human suffering from OA, comprising administering to the human a therapeutically effective amount of rAAV, wherein the rAAV encodes lubricin ligated to actuate on a promoter. The method described above comprising an AAV vector genome comprising a nucleic acid.
31. Lubricin The nucleic acid encoding the polypeptide has at least 90% identity with the sequence set forth in SEQ ID NO: 6, or the nucleic acid has an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 7. Code, the method described in any of the items 26-30.
32. The method of any of items 26-30, wherein rubricin has the amino acid sequence set forth in SEQ ID NO: 7.
33. The promoters are CMV IE promoter, RSV promoter, HSV-1 TK promoter, SV40 early promoter, SV40 late promoter, phosphoglycerate kinase gene promoter, metallothioneine gene promoter, α-1 antitrypsin gene promoter, albumin gene promoter, collagenase gene promoter. , Elastase I gene promoter, CBA promoter, β-actin gene promoter, β-globin gene promoter, γ-globin gene promoter, α-fetoprotein gene promoter, and muscle creatin kinase gene promoter. The method described in any of 32.
34. The method according to any of items 19-25, wherein the rAAV comprises the rAAV vector genome encoded by the plasmid Ps-AAV-ITR / CBA-cHAS2co-BGH.
35. The method according to any of items 26-32, wherein rAAV comprises the rAAV vector genome encoded by the plasmid Ps-AAV-ITR / minCBA-HI-cLub1co-BGH.
36. A method of preventing the development of OA in a subject mammal at risk, comprising administering to a dog a therapeutically effective amount of rAAV, wherein the rAAV is ligated to actuate on a promoter. The method comprising an rAAV vector genome comprising a nucleic acid encoding HAS2.
37. The nucleic acid encoding the HAS2 polypeptide encodes HAS2 having at least 90% identity with the sequence set forth in SEQ ID NO: 2, or at least 90% identity with the amino acid sequence of SEQ ID NO: 2. The method described in item 36.
38. 36 or 37. The method of item 36 or 37, wherein the HAS2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 3.
39. A method of preventing the development of OA in a subject mammal at risk, comprising administering to a dog a therapeutically effective amount of rAAV, wherein the rAAV is ligated to actuate on a promoter. The method comprising an rAAV vector genome comprising a nucleic acid encoding a rubricin.
40. The nucleic acid encoding lubricin has at least 90% identity with the sequence set forth in SEQ ID NO: 6, or the nucleic acid has an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 7. Code, the method described in item 39.
41. 40. The method of item 40, wherein the rubricin polypeptide has the amino acid sequence set forth in SEQ ID NO: 7.
42. The promoters are CMV IE promoter, RSV promoter, HSV-1 TK promoter, SV40 early promoter, SV40 late promoter, phosphoglycerate kinase gene promoter, metallothioneine gene promoter, α-1 antitrypsin gene promoter, albumin gene promoter, collagenase gene promoter. , Elastase I gene promoter, CBA promoter, β-actin gene promoter, β-globin gene promoter, γ-globin gene promoter, α-fetoprotein gene promoter, and muscle creatin kinase gene promoter. The method described in any of 38.
43. 26. The method of item 26, wherein the rAAV comprises the rAAV vector genome encoded by the plasmid Ps-AAV-ITR / CBA-cHAS2co-BGH.
44. 26. The method of item 26, wherein rAAV comprises the rAAV vector genome encoded by the plasmid Ps-AAV-ITR / minCBA-HI-cLub1co-BGH.
45. The method of any of items 19-44, wherein rAAV is administered intra-articularly.
46. A recombinant plasmid vector containing a nucleic acid sequence encoding a canine HAS2 polypeptide ligated to actuate on a promoter.
47. The nucleic acid sequence encoding the HAS2 polypeptide has at least 90% identity with the sequence set forth in SEQ ID NO: 3, or the nucleic acid has an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 2. 46. The recombinant plasmid according to item 46, which encodes a HAS2 polypeptide comprising.
48. The recombinant plasmid according to item 46 or 47, wherein the HAS2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2.
49. The recombinant plasmid according to any of items 46-49, comprising pCBA-HI-cHAS2-BGHpA.
50. A recombinant plasmid vector containing a nucleic acid sequence encoding a shortened inulbricin ligated to act on a promoter.
51. The nucleic acid sequence encoding lubricin has at least 90% identity with the nucleotide sequence set forth in SEQ ID NO: 6, or the nucleic acid has an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 7. The recombinant plasmid according to item 50, which encodes a rubricin containing.
52. The recombinant plasmid according to item 50 or 51, wherein the rubricin polypeptide has the amino acid sequence set forth in SEQ ID NO: 7.
53. The promoters are CMV IE promoter, RSV promoter, HSV-1 TK promoter, SV40 early promoter, SV40 late promoter, phosphoglycerate kinase gene promoter, metallothioneine gene promoter, α-1 antitrypsin gene promoter, albumin gene promoter, collagenase gene promoter. , Elastase I gene promoter, CBA promoter, β-actin gene promoter, β-globin gene promoter, γ-globin gene promoter, α-fetoprotein gene promoter, and muscle creatin kinase gene promoter. The recombinant promoter according to any one of 49 or 50-52.
54. Recombinant AAV5 viral vector comprising the nucleotide sequence set forth in SEQ ID NO: 8.
55. 55. An rAAV comprising the rAAV vector according to item 53.
56. A pharmaceutical composition comprising the rAAV of item 55 and at least one pharmaceutically or veterinarily acceptable carrier, excipient or vehicle.
57. A method for treating a target mammal suffering from osteoarthritis, which comprises the step of intra-articular administration of a therapeutically effective amount of the pharmaceutical composition according to item 56 to the target mammal.
58. 58. The method of item 57, wherein the subject mammal is a human or dog.
59. Adeno-associated virus (AAV) -based biological delivery and expression system used in the treatment of OA in mammalian joints by long-term gene expression of HAS2 or rubricin in synovial and / or cartilage cells. , Said system comprising rAAV, where the rAAV comprises a nucleic acid sequence encoding HAS2 or rubricin, an rAAV vector containing left and right AAV reverse terminal repeats (LITR and RITR), synovial cells and / or. Expression of the HAS2 or lubricin gene in cartilage cells is regulated by a promoter, which is located upstream of the leading frame of the nucleic acid sequence encoding HAS2 or lubricin, and the promoter is specifically directed by increased levels of immunostimulators. The system to be activated.
60. The AAV system according to item 59, wherein HAS2 is mammalian HAS2.
61. The AAV system according to item 59 or 60, wherein HAS2 is human HAS2.
62. The AAV system according to any of items 59-61, wherein the promoter is an inflammation-inducing promoter.
63. Inflammation-inducing promoters include NF-KB promoter, interleukin 6 (IL-6) promoter, interleukin-1 (IL-1) promoter, tumor necrosis factor (TNF) promoter, cyclooxygenase 2 (COX-2) promoter, and complement. Item 62. The AAV system according to item 62, which is selected from the body factor 3 (C3) promoter, the serum amyloid A3 (SAA3) promoter, the macrophage inflammatory protein-1a (MIP-1a) promoter and the hybrid construct described above.
64. In any of items 59-63, the rAAV vector genome comprises HAS2 comprising the amino acid sequence of SEQ ID NO: 2, rubricin comprising the amino acid sequence of SEQ ID NO: 7, or a nucleic acid encoding the functional variant described above. The described AAV system.
65. The AAV system according to any of items 59-64, wherein the rAAV vector genome comprises a marker gene that allows the vector genome to be monitored in synovial and / or chondrocytes.
66. The AAV system according to any of items 59-65, wherein the rAAV vector genome comprises a nucleic acid having at least 80% or 90% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 6.
67. The AAV system according to any of items 59-66, wherein the rAAV vector genome comprises the nucleic acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 6.
68. The AAV system according to any of items 59-67 for the treatment or prevention of osteoarthritis.
69. A pharmaceutical composition comprising the AAV system according to any of items 59-68.
70. The rAAV comprising an rAAV vector, said rAAV, comprising a nucleic acid sequence encoding a canine HAS2 polypeptide ligated to actuate on a promoter.
71. The nucleic acid sequence encoding the HAS2 polypeptide has at least 90% identity with the sequence set forth in SEQ ID NO: 3, or the nucleic acid has an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 2. The rAAV of item 70, which encodes a HAS2 polypeptide comprising.
72. The rAAV of item 70 or 71, wherein the HAS2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2.
73. An rAAV containing an rAAV vector containing a nucleic acid sequence encoding an inulbricin ligated to actuate on a promoter.
74. The nucleic acid sequence encoding lubricin has at least 90% identity with the nucleotide sequence set forth in SEQ ID NO: 6, or the nucleic acid has an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 7. The rAAV of item 73, which encodes a rubricin containing.
75. The rAAV of item 73 or 74, wherein the rubricin polypeptide has the amino acid sequence set forth in SEQ ID NO: 7.
76. AAV according to any of items 50-53, wherein the rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO: 8.
77. The promoters are CMV IE promoter, RSV promoter, HSV-1 TK promoter, SV40 early promoter, SV40 late promoter, phosphoglycerate kinase gene promoter, metallothioneine gene promoter, α-1 antitrypsin gene promoter, albumin gene promoter, collagenase gene promoter. , Elastase I gene promoter, CBA promoter, β-actin gene promoter, β-globin gene promoter, γ-globin gene promoter, α-fetoprotein gene promoter, and muscle creatin kinase gene promoter. The rAAV described in any of 72 or 74-76.
78. The rAAV according to any of items 71-77, comprising the AAV2 capsid or the AAV5 capsid.
79. A pharmaceutical composition comprising rAAV according to any of items 71-78, and at least one pharmaceutically or veterinarily acceptable carrier, excipient or vehicle.
80. A method for treating a target mammal suffering from osteoarthritis, which comprises the step of intra-articular administration of a therapeutically effective amount of the pharmaceutical composition according to item 79 to the target mammal.
81. 80. The method of item 80, wherein the subject mammal is a human or dog.
82. Isolated nucleic acid having the sequence shown in SEQ ID NO: 4.
83. Isolated polypeptide having the sequence set forth in SEQ ID NO: 5.
The present invention will now be represented in accordance with the following non-limiting claims.

Claims (15)

A rAAV comprising a recombinant adeno-associated virus (rAAV) vector, said rAAV comprising a nucleic acid sequence encoding a canine HAS2 polypeptide ligated so that the rAAV vector can act on a promoter. The nucleic acid sequence encoding the HAS2 polypeptide has at least 90% identity with the sequence set forth in SEQ ID NO: 3, or the nucleic acid encoding the HAS2 polypeptide has at least 90% identity with the amino acid sequence set forth in SEQ ID NO: 2. The HAS2 polypeptide comprising the amino acid sequence of, or the HAS2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2, and / or the recombinant plasmid comprises pCBA-HI-cHAS2-BGHpA, claim 1. RAAV described in. The rAAV comprising an rAAV vector, wherein the rAAV vector comprises a nucleic acid sequence encoding a shortened inulbricin ligated to actuate on a promoter. The nucleic acid sequence encoding lubricin has at least 90% identity with the nucleotide sequence set forth in SEQ ID NO: 6, or the nucleic acid contains an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 7. The rAAV according to claim 3, which encodes. The rAAV of claim 3 or 4, wherein the rubricin polypeptide has the amino acid sequence set forth in SEQ ID NO: 7. The rAAV according to any one of claims 3 to 5, wherein the rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO: 8. The promoters are CMV IE promoter, RSV promoter, HSV-1 TK promoter, SV40 early promoter, SV40 late promoter, phosphoglycerate kinase gene promoter, metallothioneine gene promoter, α-1 antitrypsin gene promoter, albumin gene promoter, collagenase gene promoter. , Elastase I gene promoter, CBA promoter, β-actin gene promoter, β-globin gene promoter, γ-globin gene promoter, α-fetoprotein gene promoter, and muscle creatine kinase gene promoter. RAAV described in any of the sections from 6. The rAAV according to any one of claims 1 to 7, wherein the rAAV comprises an AAV2 capsid or an AAV5 capsid. The rAAV of claim 8, wherein the rAAV comprises an AAV5 capsid. A pharmaceutical composition comprising the rAAV of any of claims 1-9 and optionally at least one pharmaceutically or veterinarily acceptable carrier, excipient or vehicle. A method of treating a subject mammal suffering from osteoarthritis (OA), comprising intra-articular administration of a therapeutically effective amount of recombinant adeno-associated virus (rAAV) to the subject mammal. The recombinant adeno-associated virus (rAAV) comprises a nucleic acid encoding a bone-protective or bone-regenerating polypeptide linked operably to a promoter, the polypeptide causing OA symptoms in the subject mammal. Expressed in vivo in an amount effective for alleviation, where the polypeptide is hyaluronic acid synthase (HAS), lubricin, interleukin-1 receptor (IL-1R) antagonist, insulin-like growth factor 1 (IGF). -1), Fibroblast Growth Factor 2 (FGF-2), Transformed Growth Factor Beta 1 (TGFβ1), Bone Forming Protein 7 (BMP7), Glucosamine-Fructose-6-phosphate Aminotransferase (GFAT), Interleukin 10 (IL-10), hemoxygenase-1 (HO-1), biologically active abbreviations thereof, or combinations thereof, said method. 11. The method of claim 11, wherein the polypeptide is a HAS2 polypeptide. 11. Or the method according to 12. The method of any of claims 11-13, wherein the HAS2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2. The nucleic acid encoding the HAS2 polypeptide has a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 3, and the rAAV is from 5'to 3'to the following element: 5'. Claims 11-14 comprising an rAAV vector genome comprising AAV reverse end repeat (ITR), stuffer nucleic acid, promoter, intron (IN), cHAS2 codon-optimized cDNA, polyadenylation signal (pA), and 3'AAV ITR. The method described in any of the sections.

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